1Departments of Obstetrics and Gynecology, 2Canadian Institutes of Health Research Group in Fetal and Neonatal Health and Development, and 3Department of Chemistry, University of Western Ontario, London N6A 5A5; 6Department of Pathology, St. Joseph's Health Centre, London, Ontario N6A 4V2; 7Department of Physiology and Biophysics, University of Calgary, Calgary, Alberta T2N 4N1, Canada; 4Departamento de Bioquímica, Instituto Superior de Ciencias Médicas de la Havana-Instituto de Ciencias Básicas y Preclínicas Victoria de Girón, Havana, Cuba; and 5Department of Chemistry, Behala College, Calcutta 700 060, West Bengal, India
Submitted 25 November 2003 ; accepted in final form 16 July 2004
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ABSTRACT |
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acute respiratory distress syndrome; captive bubble tensiometer; electron microscopy; surface tension; surfactant biophysical impairment
It is well recognized that surfactant is critical for normal lung function. For example, surfactant deficiency is considered a major contributing factor to the neonatal respiratory distress syndrome (RDS), and surfactant supplementation has proven highly beneficial with this condition (40). Furthermore, alterations in surfactant levels, composition, and activity are associated with acute lung injury (ALI) and the acute respiratory distress syndrome (ARDS) (9, 11, 20). Numerous animal studies have demonstrated that surfactant treatment can moderate or mitigate ALI induced by a variety of mechanisms. However, intervention with human clinical disease has not been as successful as animal experimentation, possibly because surfactant is not as efficacious with established pulmonary dysfunction.
Although the mechanisms involved in surfactant dysfunction with ALI and ARDS are multifactorial, it seems likely that inhibition of surfactant activity by nonsurfactant serum proteins accounts for a significant part of the deleterious effects on lung function (16, 20). Serum protein influx into the air space occurs during ALI and ARDS as a result of increased capillary permeability. However, the mechanism by which serum proteins inhibit surfactant function is not understood.
The present report describes the effect of two PC-binding proteins, C-reactive protein (CRP) and SP-A, on the surface activity of bovine lipid extract surfactant (BLES) (58, 61). BLES is a clinically approved surfactant used to treat neonatal respiratory distress in Canada. BLES contains surfactant phospholipids, mainly PC, 80%, and PG,
12%, plus SP-B and SP-C. BLES is depleted of the neutral lipid cholesterol and of the water-soluble surfactant proteins SP-A and SP-D. SP-A (and SP-D) is a member of the collagen-like lectin (collectin) family of proteins that function in the host defense antimicrobial immune system (23).
CRP is an acute-phase reactant produced by the liver in response to cytokines and is used clinically as a general marker of inflammation (50). CRP is found in elevated levels in the blood of patients with various diseases, including pneumococcal infections, RDS, and ARDS (12, 17, 18). CRP is elevated in the bronchoalveolar lavage (BAL) fluids in lung injury and lung transplantation (4). Very high levels of CRP in BAL have been reported with ARDS, where it can account for 10% of total BAL proteins (21). CRP is a 120-kDa pentamer of five identical subunits noncovalently assembled into a planar ring that belongs to the pentraxin family (47, 48). Although the crystal structure of this protein has been known for some time, its physiological functions are not completely clear (26, 39, 54). CRP was first identified through its capacity to bind to phosphorylcholine groups in C-polysaccharides of the cell walls of pneumococcal bacteria in a calcium-dependent manner, which leads to agglutination of the bacteria and activation of the complement cascade (48). It has been shown that this protein binds free phosphorylcholine and PC or DPPC in vesicles (39, 54). Although normally produced by the liver, CRP is also expressed in small amounts by alveolar macrophages, for example, with lipopolysaccharide stimulation (8). Entry of CRP into the lung with ARDS probably also occurs through leakage of plasma proteins in edema fluid. Several previous in vitro studies have also shown that CRP impairs surfactant adsorption (1, 4, 21, 24).
In contrast to CRP, the role of SP-A in surfactant function has been well studied (22, 31, 33). SP-A enhances adsorption of lipid extract and model surfactant samples containing SP-B in a calcium-dependent manner. SP-A and SP-B are critical constituents of tubular myelin. Addition of SP-A to lipid extract or model surfactants containing SP-B greatly reduces the compressibility of surfactant films, resulting in a more effective decrease in during surface area reduction (59). SP-A increases the surface activity of surfactant preparations at low calcium levels and can block the inhibitory effects of serum proteins (6, 7, 53). The manner by which SP-A has these beneficial effects on surfactant function is not understood.
It has been reported that CRP interferes with surfactant adsorption (4, 21, 24) and increases the minimum surface tension during pulsation on a pulsating bubble machine (1). However, the effect of this protein on surfactant function has not been studied extensively. Furthermore, the effect of SP-A on CRP-mediated inhibition has not been examined. The present study compares the effects of these proteins on surface activity of BLES using a captive bubble tensiometer (CBT) to monitor film formation and the ability of adsorbed films to adsorb films to achieve low s during quasistatic and dynamic surface area cycling. Interestingly, it was found that the addition of SP-A blocked the ability of CRP to inhibit BLES. These studies provide further evidence for the ability of SP-A to improve surfactant function and could provide insights relevant to improving lung function in ALI and ARDS.
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MATERIALS AND METHODS |
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Captive bubble tensiometry.
All experiments were performed on a custom-built CBT whose design has been described elsewhere (28, 42, 4446). Briefly, the CBT contains a circular glass chamber with an agar plug and a plunger and a camera to video record images of the captive bubble in the chamber (28, 41). The chamber is filled with the suspension to be tested, and after equilibrating the solution to 37°C, an air-bubble of 34 mm in diameter is drawn into the suspension by moving the plunger upward. Video recording of the bubble in the chamber is continuously performed, and the change of bubble shape over time is analyzed to give the as a function of time for adsorption isotherms. After initial adsorption, the chamber is sealed, and quasistatic or dynamic compression-expansion of the adsorbed films in the bubble is initiated. The quasistatic cycling is performed by moving the plunger to produce
10% bubble volume reduction per step. After a brief period of
10 s for equilibrium, the reduction step is repeated. Once minimum
is achieved, the bubble is allowed to rest for 11.5 min, and then the volume is increased stepwise until the original surface area is attained.
For dynamic cycling, the adsorbed films are cycled at a rate of 30 cycles/min continuously, and the bubble images are video recorded (28). The recorded images are digitized and analyzed using a custom-designed image analysis system that measures the minimum and maximum diameter of each bubble at various volumes to determine . The bubble area vs.
isotherms were plotted from these measurements (42). Emulsions of BLES (50 µg/ml), BLES (50 µg/ml) plus CRP (25 µg/ml), and BLES (50 µg/ml) plus SP-A (0.5 µg/ml) were incubated at 37°C for 20 min and were studied in the CBT for adsorption and dynamic cycling. The BLES phospholipid to CRP ratio was
60:1 (mol:mol) as described in a previous study (24). In some cases, 10 mM phosphorylcholine chloride were included with the emulsions studied.
Electron microscopy.
Small amounts of the BLES plus protein (SP-A and CRP; 500 µg) emulsions were removed from the diluted stocks in buffer A just before performing captive bubble studies for morphometry using transmission electron microscopy (TEM). The samples were immediately fixed using glutaraldehyde, pelleted, and processed using methods similar to those described previously (27). The pellets were postfixed with potassium ferrocyanide-reduced osmium tetroxide, embedded in London Resin-White (Polyscience, Warrington, PA), and dehydrated in an acetone series by progressive lowering of temperature. The samples were infiltrated in resin at room temperature and polymerized at 60°C. Ultrathin sections (
90 nm) of these embedded samples were cut using diamond knives and stained with uranyl acetate and lead citrate. The grids were imaged using a Philips 410 transmission electron microscope, and micrographs were obtained at x14,700 magnification (27).
Electrospray ionization mass spectrometry. Mass spectrometry (MS) was performed on the chloroform:methanol extracts of BLES (3). Electrospray ionization mass spectrometry (ESIMS) was performed on a SCIEX triple quadrapole (model PI 365; SCIEX, Concord, ON, Canada) by adding 0.01 N NaOH (in methanol:water, 9:1) to the organic extracts and collecting the spectra from mass/charge (m/z) units of 1001,000 by methods similar to those used by Postle et al. (35, 37). This procedure allows for observation of the relative abundance of single charged phospholipid species as discussed elsewhere (35, 52). In addition, some studies on BLES organic extracts were conducted by Dr. A. Postle using MS:MS fragmentation, and liberated phosphorylcholine groups were used to confirm the nature of parent compounds as PC species as described elsewhere (35, 37).
Statistical analysis.
The compression-expansion isotherms were analyzed as discussed previously (45, 46). Briefly, the compression isotherms from three or more independent bubble studies were plotted as separate -bubble area profiles (28). The values were averaged, and the mean and SE were plotted to display the isotherms in the figures to describe deviations in surface area for the independent experiments. Statistical comparisons of the data in Table 1 and Fig. 5 were performed using SPSS Base 9 program (SPSS, Chicago, IL). Differences were compared by analysis of variance with Tukey's post hoc test. The compressibilities of the films at a
of 15 mN/m [C15 = 1/A (
A/
)], where A is surface area and
A/
is the slope at a
of 15 mN/m for each surfactant, were compared against a baseline value of 0.005 mN/m, the compressibility of pure DPPC films (42), using a t-test. P values of <0.05 were considered significant.
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RESULTS |
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Addition of SP-A to BLES (Fig. 4A) led to a modest decrease in the compression required to achieve min
0 mN/m during the first cycle. Surface tension remained closer to equilibrium during expansion. This results in a decreased hysteresis compared with BLES alone (Fig. 3A). By the fourth compression (Fig. 4A), film compressibility was improved such that
15% surface area reduction was sufficient to achieve
min. In addition, hysteresis was virtually abolished.
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The effects of CRP on the surface properties of BLES during dynamic surface area cycling during the first, tenth, and twentieth cycles are depicted in Fig. 5. Dynamic compression of adsorbed BLES films results in an initial sharp decrease in , followed by a more moderate fall, so that
mins of
1 mN/m are reached with
50% surface area reduction. Film expansion results in a sharp increase in
such that maximum
(
max) values of
45 mN/m are reached at the original bubble size. By the tenth compression,
mins near zero are attained with
40% surface area reduction despite being initiated at 4045 mN/m.
continues to increase rapidly during film expansion. Similar overall characteristics are observed during the twentieth dynamic cycle except that hysteresis is clearly diminished.
As in the case of quasistatic cycling, addition of CRP markedly limits the ability of the films to attain low during dynamic film compression (Fig. 5B). With succeeding compression:expansion cycles,
max increases to >50 mN/m. There is no improvement in
min with cycling. Addition of SP-A to BLES:CRP samples (Fig. 5C) results in a marked improvement in surface activity such that the compressed films are capable of attaining low
mins with compression ratios similar to those required for BLES alone. Furthermore,
max is reduced compared with BLES alone. As a result, the BLES:CRP:SP-A curves show somewhat lower hysteresis compared with surfactant alone.
Table 1 compares the adsorption rates, mins, and C15 values for the BLES plus protein systems studied in the CBT. The
of the mixtures analyzed between 30 s and 10 min are not significantly different among the BLES and BLES plus CRP or SP-A. However, the adsorption rates calculated at 90 s are significantly different between the BLES plus CRP system compared with BLES alone. This shows that CRP can significantly inhibit the adsorption rate of bovine surfactant extract, and either phosphorylcholine or SP-A can reverse this inhibition. The
min values obtained during quasistatic compression are also significantly different between the BLES and BLES plus CRP, showing that the BLES plus CRP films cannot achieve near 0
even at maximal compression. The C15 values were calculated to estimate the compressibility in the fourth quasistatic cycle. This allows for a direct comparison of the film compressibility values relative to those of pure DPPC (C15 = 0.005) as published previously (34, 45, 46). The C15 values for BLES and BLES plus phosphorylcholine (
0.008) are slightly higher than those for DPPC (0.005). However, the C15 value for the BLES plus CRP film is 30 times higher and significantly different from those of BLES alone or DPPC. This shows that films with CRP are highly compressible, but phosphorylcholine or SP-A restores the compressibility of BLES plus CRP films so that the values approach those for DPPC. These data suggest that films formed with CRP act as if SP-B and SP-C were not present. Addition of phosphorylcholine or SP-A reverses the effects of CRP.
In addition to alterations in compressibility, SP-A and CRP also influence the surface area reduction that is required to reduce to low values. It will be noted that, due to the variable elevations in
max during compression:expansion cycling, film compressibility cannot be directly compared in Figs. 3 and 4. Therefore, the percentage surface area compressions required to reduce
from 20 mN/m to 1 mN/m were determined (data not shown). In some cases, for example, BLES:CRP, where low
s are not reached, this required extrapolation of the curves. Comparison of the area reductions required revealed that with both quasistatic (Figs. 35) and dynamic (Fig. 5) cycling, compressibility decreased during cycling. Addition of CRP significantly (P < 0.05) increased the calculated area reductions required for the initial compression. In fact, these calculated values show that, in the presence of CRP,
near zero could not be attained, even with 100% surface area reduction. Addition of phosphorylcholine or SP-A reduced the percent compression required to values similar to those observed in the absence of CRP.
The manner in which CRP and SP-A interact with BLES was further investigated by TEM, as illustrated in Fig. 6. The TEM images of BLES show large multilamellar vesicular (MLV) structures 810 µm in diameter, where the lamellae (lipid bilayers) are very tightly packed. However, single bilayers are difficult to identify due to the tight packing of the lamellae. Addition of CRP to this system did not significantly alter these MLV structures, except that the large size of the vesicles tended to be reduced in diameter. However, the structure of BLES is drastically altered in the systems containing SP-A with or without CRP. As shown in the higher magnification TEM image of the BLES plus SP-A system, the lamellae of the MLV bodies unravel into more planar structures. In some areas, the lamellae are more distinct because they are separated by a fuzzy material, probably SP-A. Large open areas excluded by the lamellae can be observed. Some very dark, dense areas are also present. Inclusion of CRP in these systems did not significantly alter these features. The multilamellar structures closely resemble the lamellar bodies found in the alveolar lining layer after secretion (10, 38). However, lamellar bodies have defined limiting membranes that are absent in the MLVs observed here. The high-magnification image of BLES plus SP-A is consistent with the lamellae being single bilayers with a thickness of close to 0.5 nm each and having a clear spacing between the layers as seen with lamellar bodies in the lung.
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DISCUSSION |
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In agreement with previous studies using other lipid extract surfactants, addition of CRP to BLES led to a depression in the rate of adsorption to the air-liquid interface to form a surface-active film (4, 21, 24). In addition, as reported by others (24), the inhibitory effects of CRP on adsorption were abolished by including the CRP ligand, phosphorylcholine. In addition to their rapid adsorption, BLES and other lipid extract surfactant films differ from phospholipid films of similar composition by being far less compressible. BLES films readily attain s far below the equilibrium
of 23 mN/m during quasistatic or dynamic surface area cycling (42, 43). CRP inclusion markedly increased compressibility such that adsorbed BLES:CRP films were barely able to lower
below equilibrium. As with adsorption, addition of phosphorylcholine (10 mM) effectively counteracted the CRP-induced inhibition. The observation that CRP markedly inhibits surfactant adsorption and increases film compressibility is in keeping with the reduced surface activity previously observed in pulsating bubble studies where CRP was added to surfactant TA (1, 21). The BLES concentrations used in the present study were low to maximize the beneficial effects of SP-A. In other studies, we have observed that addition of CRP (25 µg/ml) to BLES at 500 µg/ml led to prolonged adsorption times, elevated
mins, and larger compression ratios to attain low
. In each case, SP-A reversed these alterations.
The manner in which CRP inhibits surfactant function is not understood. As a member of the pentraxin family, CRP forms five-member planar, doughnut-shaped rings. The five CRP protomers are in a parallel formation so that the phosphorylcholine binding sites face in a common direction relative to the plane of the ring. CRP protomers possess a large cleft located on the other side of the planar ring (opposite the phosphorylcholine sites) that is thought to be responsible for binding the complement factors C1q and FcyR antibody fragments during the induction of phagocytosis (50). A plausible working model would consider CRP binding of PC molecules to constitute an essential factor in surfactant inhibition since such inhibition can be impeded by the water-soluble ligand phosphorylcholine. CRP can bind PC-containing liposomes, low-density lipoprotein, and natural (e.g., erythrocyte) membranes. However, such binding is very low unless lyso-PC is present or the lipids are oxidized (5, 54). CRP also binds platelet-activating factor (PAF) and synthetic PC analogs in which the phosphorylcholine head group is attached to the hydrophobic moiety by a linker. These findings have led to the suggestion that CRP binding to PC requires perturbation of the surface of the monolayer or bilayer. The basis of the apparently effective binding of CRP to DPPC and/or other PC species in BLES is not known since it occurs without lipid oxidation or high levels of lysophospholipids. CRP lipid association can be enhanced by cholesterol (51), but this sterol is removed from BLES during preparation (58, 61). The ability of CRP to interact with BLES, DPPC, and/or PC may be explained by the presence of the low-molecular-weight surfactant apoproteins SP-B and SP-C, which promote phospholipid adsorption and influence surfactant structure, for example, by promoting lipid mixing (13). Nevertheless, examination of BLES:CRP vesicles by TEM did not generate evidence for significant structural alterations. The manner in which CRP interacts with BLES and alters its surface activity must be investigated further.
In addition to phosphorylcholine, the inhibitory effects of CRP on BLES adsorption and the ability to attain low s during quasistatic and dynamic compression were negated by SP-A. Although it has been appreciated that addition of SP-A to lipid extract surfactants and model surfactant preparations containing SP-B enhances adsorption rate and decreases film compressibility, the bases of these beneficial effects are not understood. In addition to CRP, SP-A counteracts surfactant inhibition by a number of other serum proteins, including albumin and fibrinogen (16, 22, 33). Very low concentrations of SP-A suffice to block protein inhibition. This would suggest that SP-A reversal of inhibition is not stoichiometrically related to inhibitor protein concentration (7). It may be that SP-A enhances surface activity of lipid extract surfactants to an extent where it is less susceptible to the inhibitory effects of nonsurfactant proteins. This could occur through an alteration in surfactant phospholipid organization. SP-A and SP-B and calcium are required for the formation of tubular myelin, as well as for blocking protein inhibition. In keeping with this suggestion, TEM of BLES:SP-A and BLES:CRP:SP-A revealed similar large loose structures that we and others (49, 55, 56) interpret as SP-A bound to the phospholipid bilayers. We consider these structures to be potential precursors of tubular myelin (30) and have previously observed tubular myelin structures when BLES was incubated with higher levels of SP-A and calcium (15). This scenario would suggest that SP-A binding of BLES bilayers somehow either protects BLES phospholipids from interacting with CRP or improves the surface activity of BLES to the extent that inhibition becomes negligible. Further studies will be required to distinguish between these and other possibilities.
CRP and SP-A share a number of properties, such as calcium-dependent binding of saturated (and unsaturated) PCs, the presence of two integrally bound calcium ions, and the formation of complex multimeric structures. As important members of the innate immune host defense system, these proteins share certain functions, such as agglutination of foreign microbes and promotion of phagocytosis by macrophages and neutrophils. CRP can coisolate with SP-A (F. McCormack, personal communication) and SP-D (8) (R. Veldhuizen, personal communication). Nevertheless, these proteins are structurally and functionally distinct. CRP can activate the classic complement cascade (50), as can mannose binding protein, a collectin highly similar to SP-A, but SP-A does not appear to share this mechanism for microbe disposal (23). However, SP-A can kill bacteria directly by increasing membrane permeabilities through an unknown mechanism (57). CRP binds phosphorylcholine and lipid-expanded fluid or oxidized phospholipids (25), whereas SP-A is highly specific for disaturated PCs (19, 29, 59). Although the X-ray crystallographic structure for CRP and the way it binds phosphorylcholine are known, the manner in which this protein binds (oxidized) PC and PAF is not known (48). The X-ray crystallographic structure for SP-A has recently been solved (14), and a putative binding site for the polar phosphorylcholine moiety of DPPC has been identified within the carbohydrate recognition domain. Furthermore, the exposed face of the carbohydrate recognition domain contains an elongated hydrophobic region that would likely account for the ability of the collectin to bind DPPC. Nevertheless, the reason that SP-A associates with surfactant systems containing SP-B in a manner that promotes rapid formation of surface films with extremely low compressibility and why such films are immune to inhibition by serum proteins such as CRP is still not clear.
In summary, our studies show that, although CRP and SP-A both bind PC, these proteins have very different effects on pulmonary surfactant. CRP has little effect on pulmonary surfactant vesicular structure but inhibits phospholipid adsorption and increases film compressibility. SP-A alters surfactant lipid morphology, enhances surfactant adsorption, and greatly reduces film compressibility. The beneficial effects of SP-A on surfactant surface activity are sufficient to completely counteract the inhibitory effects of CRP and a number of other serum proteins. The present studies could serve as an experimental platform for investigations clarifying the differences in lipid-protein interactions arising with these two proteins.
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GRANTS |
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ACKNOWLEDGMENTS |
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K. Nag and K. Rodriguez-Capote were supported in part by Canadian Lung Association-Canadian Institutes of Health Research (CIHR) Fellowships.
Present address of K. Nag: Department of Biochemistry, Memorial University, St. John's, Newfoundland, Canada A1B 3X9.
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FOOTNOTES |
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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. Section 1734 solely to indicate this fact.
* K. Nag and K. Rodriguez-Capote contributed equally to this work.
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REFERENCES |
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