1 Laboratoire de Physiologie Respiratoire, Unité de Formation et de Recherche Cochin Port-Royal, Assistance Publique-Hôpitaux de Paris Université Paris V, and 2 Service de Médecine Néonatale, Hôpital Cochin Port-Royal, 75014 Paris, France; and 3 Laboratory of Veterinary Biochemistry and Graduate School of Animal Health, 3508 TD Utrecht, The Netherlands
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ABSTRACT |
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The cellular mechanisms by which pulmonary surfactant exerts its effects, including anti-inflammatory or proinflammatory effects, have remained elusive. To address the issue of whether plasma membrane modifications represent a target for these mechanisms, we designed an experimental protocol involving the determination of changes in cAMP levels under membrane-dependent or -independent stimulatory pathways. The effects of a modified natural porcine surfactant, Curosurf, and the major surfactant protein A were evaluated on resting and stimulated cAMP levels of human monocytes. We found that agents that elevate intracellular cAMP exhibit different susceptibilities toward a preexposure to Curosurf. The rise in cAMP induced by membrane-active agents such as cholera toxin or the diterpene forskolin was significantly inhibited by monocyte preexposure to Curosurf. In contrast, the rise in cAMP induced by the membrane-permeant phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine or by the Bordetella pertussis toxin adenylate cyclase-hemolysin was unaffected by Curosurf. Surfactant protein A did not affect either cAMP levels or the inhibitory capacity of Curosurf. We suggest that a plasma membrane-associated event affecting the mechanism underlying the effects of cholera toxin or forskolin is involved in the inhibition of cAMP accumulation caused by Curosurf.
pulmonary surfactant; surfactant-associated protein A; adenosine 3',5'-cyclic monophosphate; plasma membrane
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INTRODUCTION |
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PULMONARY SURFACTANT, a complex mixture of lipids and proteins consisting of 90% lipids (mainly phospholipids) and 10% proteins, including 5% surfactant-associated proteins, lines the alveolar surface of the lung (4). Saturated phosphatidylcholine is the major surfactant phospholipid component, whereas surfactant protein (SP) A, a hydrophilic, highly glycosylated protein, is the most abundant SP (4).
Although its primary function is to reduce surface tension in the alveoli, a growing number of studies indicate that pulmonary surfactant can also influence the function of inflammatory cells (28). For example, surfactant preparations inhibit the release of proinflammatory cytokines by monocytes (21, 25) or alveolar macrophages (22). These inhibitory effects of surfactant appear to be due to its phospholipid components, and, conversely, most proinflammatory functions known to be inhibited by surfactant, including cytokine release (13, 15), oxidative responses (26), phagocytosis (23, 24), and cell adhesion properties (14), can be stimulated by SP-A. However, inhibition of proinflammatory effects has also been reported with SP-A (12).
The precise mechanism of action of pulmonary surfactant and SP-A has remained elusive. Exposed monocytes rapidly ingest surfactant material in which the lipid components are incorporated into the plasma membrane (27). It is likely that surfactant interferes with plasma membrane structures, and previous observations with electron microscopy performed by our group (17) are consistent with such a pathway in human monocytes exposed to Curosurf, the modified natural porcine surfactant.
We tested this hypothesis more specifically by measuring changes in the level of cAMP, the ubiquitous second messenger that regulates many biochemical responses normally occurring in the lung. We took advantage of the capacity of cAMP to be increased through distinct regulatory pathways either dependent or independent on a membrane-associated event. We focused on the cAMP-stimulating pathways evoked by the secretory product (cholera toxin) of Vibrio cholerae, the diterpene forskolin, and the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) as well as the exogenous adenylate cyclase-hemolysin (AC-Hly) of Bordetella pertussis. We found that Curosurf and SP-A alone or combined had no effect on baseline intracellular cAMP levels. In contrast, Curosurf alone or combined with SP-A inhibited cAMP accumulation when stimulated by membrane-dependent agonists such as cholera toxin or forskolin but not by membrane-independent agents such as IBMX or AC-Hly.
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MATERIALS AND METHODS |
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Reagents and Media
RPMI 1640 medium, L-glutamine, HEPES buffer, FCS, Dulbecco's phosphate-buffered saline (PBS), pH 7.4, and trypsin-EDTA (0.05% trypsin-0.02% EDTA) were obtained from ICN Biomedicals (Costa Mesa, CA). Ficoll-Paque was from Pharmacia (Uppsala, Sweden). Cholera toxin, forskolin, and IBMX were purchased from Sigma (St. Louis, MO). Cholera toxin was used at concentrations between 0.01 and 10 µg/ml. Stock solutions of forskolin (10 mM) were made in ethanol and kept atModified Natural Porcine Surfactant and SP-A Preparations
Curosurf was kindly provided by Serono Laboratories (Boulogne, France). This modified natural surfactant was isolated from pig lungs by chloroform-methanol extraction and liquid-gel chromatography. It contains 99% polar lipids, mainly phospholipids, with >40% dipalmitoylphosphatidylcholine and ~1% hydrophobic SPs, although the extraction procedure excludes the hydrophilic SP-A (19). SP-A was isolated from bronchoalveolar lavage fluids from patients with alveolar proteinosis as previously described (11). Briefly, surfactant proteins were isolated by butanol precipitation (1:50 vol/vol), centrifuged, and extracted in 20 mM octyl-Monocyte Isolation
Mononuclear cells were isolated from buffy coats obtained from healthy donors by Ficoll-Paque density gradient and further purified by adherence. Cells were counted in a Malassez hemacytometer and suspended at 1 × 107 cells/ml in supplemented RPMI 1640 medium. The cell suspension was plated in 35-mm plastic petri dishes (1 ml/dish; Falcon, Becton Dickinson, Lincoln Park, NJ). After 45 min of adherence at 37°C, nonadherent cells were removed by two washes with PBS. The purity of the monocyte population was determined in previous studies with flow cytometry and the fluorescent monoclonal antibody anti-CD14 (the lipopolysaccharide receptor in monocytes). At least 85% of the monocyte population reacted positive to CD14 (3).Preincubation of Monocytes With Curosurf
Long-term incubation. Adherent monolayers containing 1 × 106 cells/dish were maintained in culture for 18 h with and without Curosurf (1 mg/ml) before the addition of cAMP-stimulating agents. We chose this concentration of Curosurf because in a previous study, Walti et al. (25) found that lower concentrations had little or no effect on monocyte inflammatory functions. Because Curosurf lacks SP-A, further experiments were performed with monocytes preincubated in the presence of SP-A alone (10 or 20 µg/ml) or Curosurf (1 mg/ml) combined with SP-A (10 µg/ml). This concentration of SP-A was estimated to be equivalent to the concentration of SP-A present in native surfactant (4).Short-term incubation. Adherent cells were used after overnight (18-h) incubation in supplemented RPMI 1640 medium. Curosurf (1 mg/ml), SP-A (10 or 20 µg/ml), or Curosurf combined with SP-A (1 mg/ml and 10 µg/ml, respectively) was added to the petri dishes 3 h before the addition of cAMP-stimulating agents.
Monocyte Treatments With cAMP-Stimulating Agents
Agents that increase the intracellular concentrations of cAMP were added to control monocytes or cells preexposed for either long or short time periods to Curosurf, SP-A, or Curosurf combined with SP-A as described in Preincubation of Monocytes With Curosurf. In experiments designed to investigate whether Curosurf directly interacts with cAMP-stimulating agents, Curosurf was washed out with PBS before cAMP-stimulating agents in supplemented RPMI 1640 medium were added. The monocytes were exposed to different concentrations of cholera toxin (0.01-10 µg/ml) for 2 h, and forskolin (10 µM), IBMX (1 or 2 mM), or the bacterial AC-Hly (1-100 ng/ml) was added to the incubation medium for 30 min. At the end of incubation, the cells were detached from the petri dishes with 0.5 ml of the trypsin-EDTA solution, washed in PBS, and resuspended in 0.5 ml of Tris · HCl buffer (50 mM, pH 7.4). The cell culture supernatants were kept at 4°C for 24 h before evaluation of cell viability by measuring lactate dehydrogenase release with a kit from Boehringer Mannheim (Mannheim, Germany) according to the manufacturer's protocol.cAMP Determination
Monocyte suspensions were transferred to Eppendorf tubes and boiled for 3 min to empty the intracellular content of cAMP in the extracellular medium. The tubes were then centrifuged (12,000 g for 3 min) to produce supernatants that were kept atStatistics
The results are expressed as means ± SE and were compared by Mann-Whitney U-test. A P value < 0.05 was considered significant. ![]() |
RESULTS |
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Effects of Curosurf and/or SP-A on Baseline Intracellular cAMP Levels
We first examined in human peripheral blood monocytes the effects of Curosurf and/or SP-A on baseline cAMP levels. Neither Curosurf alone, whether after a short (3-h) or a long (18-h) incubation period, SP-A alone (3 or 18 h, 10 or 20 µg/ml), nor Curosurf combined with SP-A (1 mg/ml of Curosurf solution containing 10 µg/ml of SP-A) significantly affected the baseline levels of cAMP in human monocytes. Those cAMP levels were 11.8 ± 0.9 pmol/106 cells in untreated monocytes (n = 8 experiments), 8.3 ± 1.0 pmol/106 cells in cells pretreated for 18 h with 1 mg/ml of Curosurf (n = 8 experiments), 11.3 ± 0.8 pmol/106 cells in cells pretreated for 18 h with 10 µg/ml SP-A (n = 6 experiments), and 9.8 ± 0.6 pmol/106 cells in cells pretreated for 18 h with Curosurf combined with SP-A (n = 6 experiments).Effects of Curosurf and SP-A on Cholera Toxin-Stimulated cAMP Levels
We next examined the effects of Curosurf on stimulated intracellular cAMP levels. We used various agents that increase the intracellular concentration of cAMP through different regulatory pathways as schematically represented in Fig. 1. Exposure of human monocytes to cholera toxin for 2 h (the required incubation time for ADP-ribosylation of Gs by cholera toxin to occur) (7) induced a dose-dependent increase in cAMP concentration, which was maximal at 50-100 ng/ml of cholera toxin (data not shown). When the monocytes were preincubated with Curosurf (1 mg/ml, 3 h) before exposure to cholera toxin, the final increase in intracellular cAMP was significantly reduced (Fig. 2 shows the results with 100 ng/ml of cholera toxin). The effect of Curosurf was time dependent inasmuch as a significantly higher inhibition was obtained when the monocytes were incubated with Curosurf for 18 h compared with that at 3 h of incubation before cholera toxin addition (62% inhibition at 18 h vs. 51% at 3 h; P < 0.05; Fig. 2). Although SP-A combined with Curosurf reduced cholera toxin-stimulated cAMP levels in a manner similar to that of Curosurf alone, SP-A alone (10 or 20 µg/ml) had no effect on cholera toxin-induced intracellular cAMP levels (Fig. 2). Those values were 62.2 ± 7.9 pmol/106 cells for monocytes stimulated with cholera toxin (n = 8 experiments), 68.7 ± 8.6 pmol/106 cells for monocytes exposed to cholera toxin and preincubated for 18 h with 10 µg/ml of SP-A (n = 6 experiments), and 70.2 ± 16.1 pmol/106 cells in monocytes exposed to cholera toxin and 20 µg/ml of SP-A (n = 6 experiments). The presence of Curosurf in the culture medium at the time of cholera toxin stimulation was not required for the inhibitory effects of Curosurf to be observed. Indeed, when Curosurf was washed out after the preincubation period, the inhibitory effects observed were similar to those observed in its presence, thus precluding a direct interaction of the stimulating agents with Curosurf.
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Effects of Curosurf and SP-A on Forskolin-Stimulated cAMP Levels
The diterpene forskolin (10 µM), which increases intracellular concentrations of cAMP via direct stimulation of adenylate cyclase, induced an approximately threefold increase in intracellular cAMP levels (Fig. 3), with maximal activation occurring after 30 min (40.7 ± 5.1 and 11.1 ± 0.9 pmol cAMP/106 cells for forskolin-treated and control cells, respectively; P < 0.05; n = 6-8 experiments). Preincubation of the monocytes with Curosurf for 3 or 18 h significantly inhibited the cAMP rise induced by forskolin as depicted in Fig. 3. Although the inhibitory effect of Curosurf was higher when the monocytes were preincubated for 18 compared with 3 h, the difference was under the level of significance. Preincubation with SP-A (3 or 18 h, 10 or 20 µg/ml) did not affect forskolin-stimulated cAMP levels or the capacity of Curosurf to inhibit the increase in cAMP levels (Fig. 3). Those values were 42.1 ± 2.9 pmol/106 cells for monocytes stimulated with forskolin and preincubated for 18 h with 10 µg/ml of SP-A (n = 6 experiments) and 37.9 ± 9.8 pmol/106 cells for monocytes stimulated with forskolin and preincubated for 18 h with 20 µg/ml of SP-A (n = 6 experiments). Curosurf combined with SP-A (1 mg/ml and 10 µg/ml, respectively) resulted in a loss of capacity of forskolin to induce cAMP that was comparable to that observed with Curosurf alone (Fig. 3).
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Effects of Curosurf and SP-A on IBMX-Stimulated cAMP Levels
IBMX increases intracellular cAMP levels by a nonmembrane-dependent inhibition of phosphodiesterase. As expected, IBMX (1 or 2 mM) induced a significant increase in intracellular cAMP levels (Fig. 4 shows the values for 2 mM). In both cases, Curosurf (1 mg/ml, preincubation for 3 or 18 h) did not impair the ability of IBMX to increase intracellular cAMP levels (Fig. 4 shows values for 2 mM IBMX after 18 h of pretreatment). Preincubation with SP-A either alone or combined with Curosurf affected IBMX-stimulated cAMP levels (Fig. 4).
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Effects of Curosurf and SP-A on Bacterial AC-Hly-Stimulated cAMP Levels
The exogenous AC-Hly of Bordetella pertussis, like IBMX, also acts without interfering with the membrane. The exposure of human monocytes to AC-Hly was associated with a marked increase in intracellular cAMP levels (Fig. 5). The increase in cAMP levels was significant from 5 ng/ml, was dose dependent, and reached a plateau at 50 ng/ml (Fig. 5). Monocytes exposed to 1 mg/ml of Curosurf for either long or short periods before the addition of AC-Hly exhibited a similar increase in intracellular cAMP compared with that in control cells (Fig. 5). The lack of effect of Curosurf against AC-Hly activity could not be attributable to the higher potency of the bacterial toxin as a cAMP-stimulating agent. As depicted in Fig. 5, the response of AC-Hly was not significantly reduced by Curosurf even when the concentration of AC-Hly was carefully adjusted in an attempt to achieve submaximal induction of cAMP by this potent stimulus. Preincubation with SP-A either alone or combined with Curosurf did not affect AC-Hly-stimulated cAMP levels (data not shown).
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DISCUSSION |
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The modified natural porcine surfactant Curosurf significantly reduced the increase in intracellular cAMP observed in response to the receptor-dependent agents cholera toxin and forskolin, whereas it was ineffective with the nonmembrane-dependent cAMP-stimulating agents IBMX and bacterial AC-Hly. The inhibitory effects of Curosurf were time dependent, suggesting that it was required to be incorporated into the cells to exert its effects. These effects were not different whether Curosurf was present during exposure of the monocytes to the cAMP-stimulating agents, thus arguing against a direct scavenger-like effect of Curosurf. This conclusion is further supported by the observation that the cellular events triggered by IBMX and AC-Hly were not modified by Curosurf. In contrast, SP-A did not affect intracellular cAMP levels, whereas preincubation with Curosurf combined with SP-A resulted in the same inhibitory effects as observed with Curosurf alone. Finally, baseline levels of cAMP were not affected by any of the preincubation conditions tested. The results obtained might be of physiological significance because estimations of the surfactant phospholipid concentrations that normally surround alveolar cells are in excess (10-20 mg phospholipid/ml alveolar lining) compared with those in our in vitro conditions (1). We suggest that the differential sensitivity of the various cAMP-stimulating agents toward the inhibitory effects of Curosurf relates to differences in the specific mechanisms and regulatory pathways by which each of them stimulates cAMP.
Cholera toxin binds to a cell-surface ganglioside site and catalyzes
the ADP-ribosylation of the -subunit of certain G proteins that
regulate the activity of adenylate cyclase, which results in an
irreversible activation of the enzyme by inhibiting GTPase activity of
the
-subunit (7). Both G proteins and the catalytic unit are
membrane-associated molecules. Forskolin activates adenylate cyclase
through a unique mechanism involving both direct modulation of the
adenylate cyclase enzyme and potentiation of the classic receptor-mediated mechanism (20). Intracellular levels of cAMP are also
regulated through the rate of breakdown of cAMP by phosphodiesterase enzymes that hydrolyze cAMP to the biologically inactive AMP. Phosphodiesterase activity is exhibited by a heterogeneous group of
distinct isoenzymes affected differently by various inhibitors (5).
Among the nonselective inhibitors, IBMX is considered a reference for
potent inhibitory activity in all phosphodiesterase forms (5).
Phosphodiesterase activities have been described as membrane bound of
unknown location but also as cytosolic enzymes, particularly in human
monocytes (8). AC-Hly enters mammalian cells and converts intracellular
ATP into cAMP through an ion-conductive pathway without interfering
with a specific cell receptor (16). Thus the cAMP-stimulating effect of
IBMX and AC-Hly bypasses both the receptor- and membrane-associated
adenylate cyclase activity.
These observations suggest that a time-dependent incorporation of the lipid components of Curosurf into monocyte membranes may represent a crucial event that causes impairment of adenylate cyclase in cholera toxin- and forskolin-stimulated monocytes. This interpretation is supported by previous morphological observations showing that control monocytes have an undulating surface with membrane folds and thin microvilli compared with surfactant-treated cells that were rounded and devoid of surface structures (17, 27). Furthermore, a time-dependent gradual loss of the adherence capacity has also been described in human monocytes cultured with Curosurf (25). Other evidence supporting the observation that Curosurf exerts its effects by interfering with the plasma membrane arises from the previous observation by Walti et al. (25) on superoxide anion production: indeed, Curosurf inhibited superoxide anion production induced by the receptor-mediated bacterial extract OM-85 but not by phorbol myristate acetate, which binds directly to cytosolic protein kinase C.
In conclusion, it appears that surfactant, but not SP-A, inhibits cAMP accumulation when a membrane-controlled factor is involved. It may be inferred that the anti-inflammatory or proinflammatory effects of surfactant or surfactant components thus relate to specific monocyte membrane modifications. It is worth noting that inhibition of intracellular cAMP is not incompatible with either the anti-inflammatory or proinflammatory effects because both an increase and decrease in cAMP have been reported after challenge with inflammatory stimuli (2, 9). Our data provide a novel insight into the mechanism by which surfactant modulates the functions of monocytic cells.
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ACKNOWLEDGEMENTS |
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We are grateful to Nicole Guiso for providing adenylate cyclase-hemolysin and to Serono Laboratories (Boulogne, France) for providing Curosurf. We also thank Sarah Kreps (Harvard University, Cambridge, MA, and University of Oxford, Oxford, UK) for reading the manuscript.
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FOOTNOTES |
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This study was supported in part by Serono Laboratories.
B. S. Polla and M. Bachelet were supported by the Institut National de la Santé et de la Recherche Médicale, and F. Pinot was supported by Serono Laboratories.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: H. Walti, Service de Médecine Néonatale, Hôpital Cochin Port-Royal, 123 Blvd. de Port-Royal, 75014 Paris, France.
Received 2 February 1999; accepted in final form 29 August 1999.
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