Eur Respir J 1999; 13: 1455±1476 Printed in UK ± all rights reserved

Copyright #ERS Journals Ltd 1999 European Respiratory Journal ISSN 0903-1936

REVIEW

Pulmonary surfactant in health and human lung diseases: state of the art
M. Griese
Pulmonary surfactant in health and human lung diseases: state of the art. M. Griese. #ERS Journals Ltd 1999. ABSTRACT: Pulmonary surfactant is a complex and highly surface active material composed of lipids and proteins which is found in the fluid lining the alveolar surface of the lungs. Surfactant prevents alveolar collapse at low lung volume, and preserves bronchiolar patency during normal and forced respiration (biophysical functions). In addition, it is involved in the protection of the lungs from injuries and infections caused by inhaled particles and micro-organisms (immunological, non-biophysical functions). Pulmonary surfactant can only be harvested by lavage procedures, which may disrupt its pre-existing biophysical and biochemical micro-organization. These limitations must always be considered when interpreting ex vivo studies of pulmonary surfactant. A pathophysiological role for surfactant was first appreciated in premature infants with respiratory distress syndrome and hyaline membrane disease, a condition which is nowadays routinely treated with exogenous surfactant replacement. Biochemical surfactant abnormalities of varying degrees have been described in obstructive lung diseases (asthma, bronchiolitis, chronic obstructive pulmonary disease, and following lung transplantation), infectious and suppurative lung diseases (cystic fibrosis, pneumonia, and human immunodeficiency virus), adult respiratory distress syndrome, pulmonary oedema, other diseases specific to infants (chronic lung disease of prematurity, and surfactant protein-B deficiency), interstitial lung diseases (sarcoidosis, idiopathic pulmonary fibrosis, and hypersensitivity pneumonitis), pulmonary alveolar proteinosis, following cardiopulmonary bypass, and in smokers. For some pulmonary conditions surfactant replacement therapy is on the horizon, but for the majority much more needs to be learnt about the pathophysiological role the observed surfactant abnormalities may have. Eur Respir J 1999; 13: 1455±1476.
The Lung Research Group, Kinderpoliklinik und Kinderklinik, Dr. von Hauner Childrens' Hospital, Ludwig Maximilians University, Munich, Germany. Correspondence: M. Griese The Lung Research Group Kinderpoliklinik und Kinderklinik Dr. von Hauner Childrens' Hospital Ludwig Maximilians University Pettenkoferstrabe 8a D-80336 Munchen È Germany Fax: 49 8951603477 Keywords: Phosholipids surface activity surfactant protein-A surfactant protein-B surfactant protein-C surfactant protein-D Received: July 14 1998 Accepted after revision December 23 1998 This work was supported by grants from Deutsche Forschungsgemeinschaft and the W. Sander Stiftung.

Pulmonary surfactant components and their dysfunction Pulmonary surfactant is a complex and highly surface active material composed of lipids and proteins which is found in the fluid lining the alveolar surface of the lungs. Surfactant plays a vital role in pulmonary physiology. Its major biophysical functions are to prevent alveolar collapse at low lung volume and to preserve bronchiolar patency during normal and forced respiration, and its major nonbiophysical, immunological, functions are the protection of the lungs from injuries and infections caused by inhaled particles and micro-organisms. A pathophysiological role for surfactant was first appreciated in premature infants with respiratory distress syndrome (RDS) and hyaline membrane disease, a condition which can nowadays be treated by means of exogenous surfactant replacement. Various other lung diseases are associated with surfactant abnormalities, and in some of these diseases replacement therapy is on the horizon. In this article, the data on the human surfactant system in health and in various disease conditions are reviewed and an overview of potential dysfunctions is given.

The composition and structure of pulmonary surfactant Pulmonary surfactant is heterogeneous with respect to biochemical composition, morphological organization and specific biophysical functions [1]. Biochemically, pulmonary surfactant is composed of approximately 90% lipid and 10% protein, the latter representing the four surfactant-associated proteins surfactant protein (SP)-A, SPB, SP-C and SP-D, as well as a large number of other, mostly serum-derived, proteins. A schematic illustration of these components and their relative sizes is given in figure 1. The majority of pulmonary surfactant lipids are phospholipids. The most abundant phospholipid, phosphatidylcholine, is largely disaturated dipalmitoylphosphatidylcholine (65%), which plays an essential role in decreasing surface tension. Pulmonary surfactant also contains a relatively large portion of phosphatidylglycerol. Studies suggest that, in surfactant, phosphatidylglycerol can be replaced by another negatively charged phospholipid, namely phosphatidylinositol, without affecting the surfactant's properties of lowering the surface tension at the air± water interface from ,70 mN.m-1 at a pure water±air

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SP-D

PL SP-A SP-B SP-C 50 nm
Fig. 1. ± Schematic structure and relative size of the principal components of pulmonary surfactant. The phospholipids (PL) are shown in the form of a bilayer, omitting the various other structural forms of organization demonstrated by electron microscopy, e.g. vesicles, sheets, tubular myelin, and lamellar bodies. Surfactant protein (SP)-A is shown as an octadecamer with a bouquet-like appearance, and SP-D is depicted as a dodecamer with a cross-like structure. The carbohydrate recognition domains of these two proteins are shown as globular formations. SP-B is shown as a monomer, although a significant amount of dimers are present in the airspaces. SP-C is depicted as an integral membrane monomer. PL: phospholipids.

interface to approximately 0±1 mN.m-1 during expiratory compression [1]. Little is known about the role of the other lipid components, of which cholesterol is the most abundant (approximately 10% by mass), the other neutral lipids occurring in trace amounts only. The most abundant SP by weight is SP-A. The SP-A monomer (molecular weight approximately 32 kDa) is a glycoprotein with three distinct structural domains [2, 3]. A long stretched collagenous domain is connected via a linking region (possibly responsible for the binding of phospholipids) to a globular region. This region contains a calcium-dependent carbohydrate recognition domain (CRD) which is able to bind both lipids and type II cells, as well as other structures (e.g. surfaces of micro-organisms). A complex oligosaccharide is also attached to this region of the SP-A molecule. Because of their mixed collagen-like and globular structure, such molecules are called collectins. The fully processed and secreted form of SP-A consists of 18 SP-A monomers (octadecamer or six trimers), organized by means of covalent disulphide bridges and noncovalent interactions in the shape of a bouquet of tulips (fig. 1). The two genes for human SP-A are located on chromosome 10 and are expressed in alveolar type II cells, bronchiolar Clara cells and airway submucosal gland cells. SP-A and SP-B (see below) both have a role in the conversion of endogenous surfactant into tubular myelin. SP-A accelerates the adsorption of surfactant phospholipids at the air±water interface, stimulates the defence system which depends on macrophages [4], reduces the inhibitory effect on surface activity of the nonsurfactant proteins within the alveolar space and possibly plays a role in the regulation of surfactant homeo-

stasis, since it inhibits surfactant secretion and increases the uptake of surfactant by type II pneumocytes [5, 6]. SP-D is the second hydrophilic surfactant protein and also a collectin [3, 4, 7]. The collagen-like domain of SPD is much larger than that of SP-A and is attached directly, without a connecting region, to the CRD domain. The molecular weight of the SP-D monomer is approximately 43 kDa. The native SP-D found in the lungs consists of 12 SP-D monomers, three of which are joined to form a trimer. Four trimers form a cross-shaped molecule (fig. 1), as demonstrated by electron-microscopic investigations. This cross-like structure (width of the molecule approximately 92 nm) may bind to bacterial lipopolysaccharide (LPS) and to cell surfaces, forming larger networks of cells or bacteria. In addition, a receptor which binds SP-D independent of its CRD domain has recently been identified on alveolar macrophages [8]. SPD is also expressed in type II cells and in Clara cells, the gene being located on chromosome 10. The majority (70%) of SP-D is found dissolved in the watery surfactant residue, whereas SP-A, SP-B and SP-C are almost entirely found in association with lipids. SP-D is able to bind phosphatidylinositol and ceramides but not much is known about its influence on the regulation of surfactant homeostasis. Recently, however, disturbances of surfactant metabolism have been reported in SP-D knock-out mice. SP-D does not play a role in the biophysical functions of surfactant. Intra-alveolar SP-B is a hydrophobic, positively charged molecule with a molecular weight of approximately 8 kDa. SP-B is coded for by a gene on chromosome 2, which is expressed in the lung by type II cells and Clara cells. A large preprotein is processed intracellularly to form the active SP-B molecule (fig. 1). SP-B is found mainly in the form of a dimer in the alveolar space, with two SP-B molecules linked to each other via disulphide bonds. The main function of SP-B is to accelerate the formation of a surface active film composed of phospholipids at the air± water interface by means of an increase in the adsorption rate by a factor of >150. This effect is further accelerated by the presence of calcium ions such that mixtures of phospholipids and SP-B display almost the same biophysical properties as whole lung surfactant. SP-B in conjunction with SP-A and calcium ions is also involved in the formation of tubular myelin. SP-A is found at every vertex of the lattice structure of these aggregates and determines the distance by which the lipid lamellae which are associated with SP-B are separated. SP-C is the only surfactant protein which is expressed exclusively by type II cells in the mature lung. The human gene is found on chromosome 8 and SP-C, too, is translated as a larger preprotein and processed intracellularly. The active molecule is a very hydrophobic polypeptide to which two palmitoyl groups are attached via covalent bonds (molecular weight 4 kDa) (fig. 1). The main function of SP-C is to maintain the biophysical surface activity of the lipids. This occurs through an acceleration of the rate of adsorption at the air±water interface as well as through an increase in the resistance of surfactant to inhibition by serum proteins or by oedema fluid. SP-B and SP-C also increase the uptake of phospholipids into type II pneumocytes. SP-C stabilizes the surface activity of the surfactant film during the expansion and compression involved in breathing.

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Biophysical functions of pulmonary surfactant (table 1) The notion that surface tension is more important than tissue elastic forces for the retractive force of the lungs at all levels of inflation was first expressed by NEERGAARD [9] in 1929. The surface tension of the alveolar air±water interface provides this retractive force opposing lung inflation. The law of Laplace illustrates that the difference in pressure between the airspace and the lining (DP) depends only on the surface tension (T) and the radius of the alveoli (DP=2 T/r). The presence of surfactant in the fluid film can lower air±water surface tensions to near zero values (table 1). This ensures that the alveolar space remains open during the whole respiratory cycle, thus preventing intrapulmonary shunts resulting in inadequate oxygenation of the blood, and this also leads to reduced work of breathing. Increasing evidence suggests that surfactant is needed not only in the alveolar part of the lung but also in the bronchioli through which air is conducted to the alveoli [10±12]. In vitro and in vivo studies have shown that a lack of surfactant leads to closure of the small cylindrical airways. In addition to this, the presence of phospholipases, proteases and exuded plasma proteins, in inflamed airways might severely disrupt the functional ability of surfactant to keep the conducting airways open [13]. Low surface tension is also important for ensuring that a net fluid flow is directed from the alveolar space into the interstitium [14]. This mechanism is of particular importance in the alveoli, because of their small diameter. In such areas, with a relatively high surface tension, a thicker fluid film may develop. Thus a well-functioning surfactant keeps the alveoli clear of liquid while also maintaining a thin fluid film. A lack of surfactant, conversely, leads to the accumulation of oedema fluid in the airspace. Lastly, pulmonary surfactant is believed to play a role in the physical removal of particulate material from the alveoli and small airways by means of the displacement of particles into the hypophase and improvement of mucociliary clearance. The molecular details of surfactant dysfunction are largely unknown. Some of the mechanisms which may lead to impaired surfactant function in pathological states are listed in table 2 and will be referred to when the individual diseases are discussed.
Table 1. ± Functions of pulmonary surfactant

The functions of surfactant in host defence The phospholipid components in large abundance under normal conditions (in neonates, phosphatidylcholine, phosphatidylglycerol and phosphatidylinositol) have been shown to suppress various lymphocyte and macrophage immune functions, whereas SP-A and SP-D have been demonstrated to activate several immune cell functions (table 1) [3, 4]. However, there is as yet no information available on the in vivo relevance of these findings. SP-A specifically interacts with alveolar macrophages and increases the intensity of their respiratory bursts, migration, chemotaxis and complement-dependent and independent phagocytosis. While SP-A stimulates the formation of cytokines and immunoglobulins by lymphocytes, the surfactant lipids inhibit lymphocyte proliferation and immunoglobulin production. SP-A binds to LPS, group A streptococci, pneumococci, Staphyloccus aureus, Mycobacterium tuberculosis, Haemophilus influenzae type A, influenza A virus, herpes simplex virus type 1, candida and Pneumocystis carinii. Specific binding of SP-A to carbohydrates such as asialo-GM2, Galactosylceramide and gp 120, amongst others also takes place [3, 4]. SP-A also binds to specific receptors on type-II cells and is probably involved in the regulation of surfactant secretion and reuptake. For SP-D there are no functions known that are related to the biophysical activity of surfactant. This molecule may be of great importance for the nonadaptive defence system of the lung. SP-D has specific binding sites on alveolar macrophages, can induce a "respiratory burst", and stimulates their phagocytotic activity. SP-D also binds to polymorphonuclear granulocytes, LPS, Escherichia coli, Pseudomonas aeruginosa, Influenza A virus and P. carinii. The precise overall roles played by SP-A and particularly SP-D in pulmonary host defence have yet to be elucidated [4]. Extracellular surfactant metabolism After synthesis by type II pneumocytes, surfactant is secreted into the alveolar space. This process of exocytosis is regulated by various stimuli [15, 16] and dependent on ontogenesis [17]. In the alveolar space and in the presence of calcium, SP-A and SP-B, the highly surface active

Biophysical functions of surfactant Prevents collapse of the alveoli and lungs during expiration Supports inspiratory opening of the lungs Prevents lung oedema formation by balancing hydrostatic filtration forces Stabilizes and keeps small airways patent Improves mucociliary transport Translocates particles 2006g should be avoided in order to prevent some of the larger aggregated forms of surfactant (e.g. tubular myelin) being lost to the cell pellet. Importantly, the lavage fluid should not be frozen before processing the cells. The lavage supernatant may be analysed as such or separated further by differential centrifugation into various fractions (fig. 2). Unfrozen material is preferred; if this is not possible, it should be indicated. A surfactant-rich pellet (LAs) is generated by centrifugation at 28,000±73,0006g. A number of groups use 40,0006g [18, 23±25]. The supernatant obtained from this centrifugation step is the SA fraction of the surfactant. A somewhat more purified surfactant fraction can be obtained by differential density gradient centrifugation [26, 27], but these methods have been used rarely for lavage samples from humans. Although not all biochemical and biophysical surfactant markers have been investigated, relatively good agreement has been demonstrated for some parameters between density gradient centrifugation and the more simple centrifugation procedures [22]. Material sampled by bronchial lavage differed in biochemical composition from that sampled by BAL, but was similar to sputum [28, 29]. The latter has also been used as

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a)

1 LA + SA Cells 2 SA LA UC SDG 3 Light Heavy Ultraheavy

must be made with great care and have to be related to their own controls. Also, BAL fluid recovery must not only be given but should also be considered when calculating the results, as this may change completely the conclusions to be drawn [33]. Reasonable technical recommendations for the lavage procedure are awaited to allow better standardization (Task Force of the European Respiratory Society). Lastly, the interpretation of BAL data in functional terms is most difficult, since the relevance of subtle changes in the quantity of alveolar lining fluid components is just beginning to be explored. Status of pulmonary surfactant in humans A large amount of data with relevance to the human lung surfactant system under normal and various pathological conditions has been collected, using approaches which differed to a greater or lesser extent with respect to patient selection and methods used. Only those studies reporting data in appropriate units, e.g. expressed per lavage volume recovered, were included in the analysis. However, in order to allow an estimate of the order of magnitude of the parameter, the means or medians of the major biochemical and biophysical surfactant parameters were collected and the means calculated (table 3). Valuable additional information not fitting into this format is given in the text. For the sake of clarity, the various diseases were grouped into certain categories, knowing that there is substantial overlap. Instead of reporting the numerical results of the individual studies, the data are summarized in table 4 by indicating the qualitative changes observed. Each symbol represents the result obtained in comparison with the appropriate control group. Healthy controls and smokers No systematic and large scale studies of the pulmonary surfactant system in healthy adults are available. However, as there is a wide range of variation even within normal subjects, each lavage study should analyse its own control groups for comparison. Most of the variation is likely to be caused by the different methods used to obtain the lavage fluids, prepare the surfactant and analyse its composition. An appropriate meta-analysis cannot be performed on these heterogeneous studies. Generally, in the lavage supernatant obtained from healthy adults phosphatidylcholine and phosphatidylglycerol together make up approximately 80% of total phospholipids and the surfactant-specific proteins represent