From Birds to Humans - ATS Journals

Translational Review From Birds to Humans New Concepts on Airways Relative to Alveolar Surfactant Wolfgang Bernhard, Patricia L. Haslam, and Joanna Floros Department of Neonatology, Faculty of Medicine, Eberhard-Karls-University, Tu¨bingen, Germany; Adult Intensive Care Unit, Royal Brompton & Harefield NHS Trust and Unit of Critical Care, National Heart & Lung Institute, Faculty of Medicine, Imperial College of Science, Technology & Medicine, Royal Brompton Hospital, London, United Kingdom; and Departments of Cellular and Molecular Physiology, Pediatrics, and Obstetrics and Gynecology, Pennsylvania State University College of Medicine, Hershey, Pennsylvania

Pulmonary surfactant is a surface-active mixture of phospholipids and specific proteins that lines the epithelial surfaces of mammalian lungs. In the alveoli, its main function is to reduce surface tension to ensure that these structures can remain open during respiratory cycles of contraction and expansion. However, surfactant is also present in the conducting airways, even though they are relatively rigid and do not need a system capable of rapidly lowering surface tension in response to compression. This has raised the question whether there is a difference in composition and function between airway and alveolar surfactant. Interest in this question has been stimulated further by the recognition that surfactant also has important functions in the immune defenses of the respiratory tract. In this review, we describe differences that have been reported between human airway and alveolar surfactant. In addition, we draw parallels between human airway surfactant and surfactant from the lungs of birds. The latter are tubular and rigid and do not undergo cycles of contraction and expansion, thus more resembling the human conducting airways than alveoli. Using this as a model, we propose a new hypothesis to explain structural and functional differences between human airway and alveolar surfactant. We suggest that the molecular composition of surfactant is adapted to differences in the architecture of pulmonary surfaces and to the dynamics of surface area changes during respiration.

Pulmonary surfactant (surface active agent) is a complex system of lipids and proteins, which lines the alveolar epithelial surfaces of the lungs of humans and other mammals (1). It plays an essential role in lung function by varying and reducing surface tension to stabilize the alveoli and prevent their collapse when the lungs undergo successive cycles of compression and expansion during breathing (2). The importance of this biophysical function is well demonstrated in conditions of alveolar surfactant deficiency, as can occur in premature infants resulting in development of respiratory distress syndrome; surfactant replacement therapy in this situation has proved of great clinical value

(Received in original form May 5, 2003 and in revised form July 7, 2003) Address correspondence to: Wolfgang Bernhard, M.D., Ph.D., Department of Neonatology, Faculty of Medicine, Eberhard-Karls-University, Calwer Straße 7, D-72076 Tu¨bingen, Germany. E-mail: wolfgang.bernhard@med. uni-tuebingen.de Abbreviations: phosphatidylcholine, PC; surfactant protein, SP. Am. J. Respir. Cell Mol. Biol. Vol. 30, pp. 6–11, 2004 DOI: 10.1165/rcmb.2003-0158TR Internet address: www.atsjournals.org

(3). Surfactant also has important biological functions that contribute in pulmonary host defense and regulation of immune and inflammatory responses (1, 4–6). However, although the properties of alveolar surfactant have been studied extensively, much less is known about the finding that surfactant is also present in the fluid that lines the conducting airways, even though the bronchi (relatively rigid and tubular) are not subject to the same dynamic stresses as the alveoli (highly “elastic” and “spherical”) (7, 8). Why then, should there be a requirement for a system capable of lowering surface tension in the conducting airways? Or is there a more general role for surfactant at airexposed pulmonary surfaces, and does the function and structure of airways versus alveolar surfactant differ in certain respects? The purpose of this article is to consider these questions and also to develop a new hypothesis regarding probable differences between airway and alveolar surfactant in humans in the light of recent knowledge about the composition and function of avian and other mammalian surfactants (9, 10). The lungs of birds are essentially rigid and tubular without alveoli (Figure 1A), and are thus more like the bronchial rather than the alveolar regions of the mammalian lung (Figure 1B) (9). Thus, we postulated that avian surfactant may show a greater resemblance to human airway rather than alveolar surfactant. In this review we advance our arguments in support of this proposal.

Surfactant Composition and Function A number of extrapulmonary structures such as the eustachian tube or stomach are lined with so-called “surfactantlike phospholipids,” but a molecular or functional similarity with lung surfactant can be ruled out (11–13). However, surfactants originating from type II pneumocytes of homeothermic vertebrate lungs are similar, although not identical to each other (8–10). Surfactants comprise ⵑ 80% phospholipids, 10% neutral lipids, and 10% proteins. Eighty percent of the phospholipids are phosphatidylcholines (PC). In addition, mammalian surfactant contains 10–15% anionic phospholipids, mainly phosphatidylglycerols (PG) (1). Although dipalmitoyl-PC (PC16:0/16:0) is the principle surface tension–lowering component, mammalian surfactants comprise ⬍ 40% of this molecule, with values down to 25% in rapidly breathing newborn rodents (10). Moreover, surfac-

Translational Review

Figure 1. Respiratory airflow in avian and mammalian lungs. Filled and open arrows denote direction of air flow during inspiration (filled arrows) and expiration (open arrows), respectively. Relative thickness of the arrows indicates the proportion of air streaming through the different areas of the respiratory system during the respiratory cycle. Dotted arrows indicate the volume changes of air sacs and alveoli of avian and mammalian lungs, respectively. In bird lungs (A ), most air directly enters the caudal air sacs during inspiration (thick black arrow), whereas a lesser part flows through the parabronchi/air capillaries into cranial air sacs (thin black arrows). During expiration the major part of inspired air streams from the reservoirs (caudal air sacs, thick open arrows) through the parabronchi/air capillaries into major distal airways, where it mixes with the deoxygenated respiratory gas stored in cranial air sacs during the inspiratory phase. Consequently, respiratory gas flow through the parabronchi, atria, and the gas-exchanging air capillaries is unidirectional and continuous during both inspiration and expiration. This principle is achieved by cranio-caudal pressure gradients in the respiratory system changing between inspiration and expiration and the consecutive opening and closing of valve systems between mesobronchi/air sacs and the parabronchi (not indicated in the figure). Hence, airflow is constant and high in the parabronchi, atria, and the gas-exchanging air capillaries, making sedimentation of small particles (filled circles) less likely. By contrast, in mammalian lungs (B) airflow is bidirectional during the respiratory cycle, with highly reduced airflow in peripheral structures, i.e., bronchioles and, particularly, the gas-exchanging alveoli. Consequently, small particles (⬍ 1 ␮m), which enter the alveoli, may sediment, making a system of first line of defense necessary, comprising alveolar macrophages, SP-A, and (phospholipid) regulators of inflammatory processes (for details see Refs. 25, 26, 27, 33, 36, 37, and 38). *SP-A is present in human bronchiolar lining fluid, but not expressed in normal (healthy) human airway epithelia (27).

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tant from cattle, which display higher respiratory rates of 60 strokes/min compared with adult humans and pigs (an average of 12–14 strokes/min), contains less PC16:0/16:0 (14). Other surfactant components are apparently “assisting components.” These include PG molecular species, unsaturated PC species, or PC species with a short fatty acid chain in the sn-2 position of the PC molecule, such as palmitoylmyristyl-PC (PC16:0/14:0), palmitoylpalmitoleoyl-PC (PC16:0/ 16:1), and palmitoyloleoyl-PC (PC16:0/18:1). These less abundant surfactant lipid components have a low gel to sol transition temperature and support surface adsorption of PC16:0/ 16:0, which is rigid at body temperature, and, therefore, cannot easily adsorb to air–liquid interfaces (15, 16). Specific proteins, namely surfactant protein (SP)-A, SP-B, SP-C, and SP-D, play an important role in alveolar surfactant homeostasis, in promoting rapid accumulation of surfactant phospholipids at the air–liquid interface, in regulation of phase separation between PC16:0/16:0 and fluid phospholipids at the interface, and in innate immune responses (1, 17). Their functions include participation in the formation of lipoprotein aggregates of “tubular myelin” by SP-A and -B in the liquid hypophase that covers alveolar cells (18), formation of lamellar bodies in type II pneumocytes and SP-C maturation by SP-B (19), promotion of phospholipid accumulation at the interface by SP-B and SP-C (17, 20), and regulation of surfactant recycling and alveolar homeostasis by SP-A and SP-D (4). In addition, SP-A and SP-D are involved in the innate host defense and/ or the regulation of inflammatory processes (4, 5). Studies of knockout mice and/or human studies (19, 21) indicate that SP-B is essential for life; the other surfactant proteins, although not essential for life, are important for surfactant biophysical function under certain conditions and/or are important for antimicrobial or other biological functions (4, 22, 23, 30). For example, potency of surfactant preparations increases dramatically in the presence of SP-A, whereas therapeutic surfactants lacking this protein achieve minimal surface tensions near zero mN/m only at concentrations 6to 12-fold higher compared with native, SP-A–containing surfactants (14, 22). Moreover, absence of SP-A causes increased susceptibility of the lungs to opportunistic bacteria such as the Pseudomonas aeruginosa (23).

Surfactant in Alveolar and Tubular Structures The above functional characteristics are a common feature of alveolar surfactant of adult mammals in general. However, there are considerable functional and biochemical differences between alveolar surfactant and surfactant in tubular structures, not only that in the conducting airways of mammalian lungs but also that in the essentially tubular avian lung (8, 9). Also, surfactant differences exist among vertebrate species that differ in respiratory rate and pulmonary maturation (10). For instance, surfactants isolated from the airways of mammals (conductive airway surfactant) and from the essentially tubular bird lung display different surface tension behavior compared with alveolar surfactant. Although alveolar surfactant easily achieves minimal surface tension values near zero mN/m upon dynamic compression of a pulsating air bubble in a surfactant suspension (which represents the situation of a lung alveolus at end expiration of the respiratory cycle),

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low surface tensions cannot be achieved with surfactant isolated from the airways of alveolar lungs or from bird surfactant (8, 9, 13). The “inferior” functional activity of ”tubular surfactant” may be explained by the Law of Laplace as it applies to tubules (P ⫽ ␥/r; P ⫽ opening pressure, ␥ ⫽ surface tension, r ⫽ radius), in contrast to its application to sphere-like structures such as the alveolus (P ⫽ 2␥/r). According to the Law of Laplace, minimal surface tensions near zero mN/m may be less critical for tubules than for alveoli. This is consistent with the observed surface tension of airways in situ, which is in the range of 20–30 mN/m (24), and with surfactant from the tubular avian lung, which does not achieve minimal surface tension values near zero mN/m within five surface area compressions by contrast with alveolar surfactant (9). Although near-zero values after prolonged cyclic compressions (more than 5 min at a rate of 20/min) cannot be ruled out (8, 9), it is clear that the extent of area compression in the alveolus differs from those in airways and in the rigid avian lung (25). However, the biochemical characteristics of conductive airway surfactant from the mammalian lungs are only partially identical with surfactant from the tubular bird lung. Although conductive airway surfactant displays a phospholipid composition similar to that of alveolar surfactant, tubular myelin is absent (7). Also, surfactant proteins can be reduced or even absent in some species (8, 26–28), which may, in most species, explain the absence of tubular myelin, as this is formed by aggregation of surfactant phospholipids with SP-A and SP-B (18). This raises the possibility that the large reservoir of surfactant provided by tubular myelin to facilitate surface film adsorption to cope with the requirements of gross changes in surface area in the alveoli during breathing is not required in the relatively rigid conducting airways. Bird surfactant, which apparently does not contain either SP-A or SP-C, has a similar or even higher concentration of SP-B than alveolar surfactant from mammals (9). Such differences may relate to morphologic and physiologic differences in the respective structures as well as to the general function of SP-B. Conductive airway surfactant appears to originate from overflow of alveolar surfactant material at the air–liquid interface (8), probably as a result of alveolar surface area compression during expiration. The comparative lack of proteins may be explained by the lack of surfactant proteins relative to phospholipids in the surface film, which may preferentially move over the ascending fluid. Moreover, surfactant proteins may be bound to epithelial cells or retained in the compartment, where they have been synthesized and secreted. Consistent with this view, expression of the SP-B gene in Clara cells close to the alveolar compartment does not compensate for a lack in SP-B expression by alveolar type II pneumocytes, first because these cells are located outside the alveoli, and second because Clara cells do not secrete lamellar bodies that contain both SP-B and lipids (29). Surfactant in birds, on the other hand, adsorbs to the interface and spreads without any surface compression of the avian rigid lung, from the type II pneumocytes of the entrances (atria) of the air capillaries into the air capillaries (9) (Figure 1A). Consequently SP-B, which is essential for rapid surface adsorption of surfactants (20), must be present both in avian “tubular”

lung and in mammalian alveolar surfactant. SP-C, on the other hand, contributes to low surface tension in small alveoli, e.g., at end-expiration, and promotes reintegration of phospholipids into the air–liquid interface after alveolar overcompression (20). No such overcompression occurs in the rigid tubular bird lung, making a physiologic need for SP-C in bird lungs unlikely. Recently, impaired lung function was demonstrated in SP-C knockout mice, suggesting the need of SP-C for surfactant function at end-expiration in vivo (30). Moreover, tubular myelin is not found in avian surfactant (9), presumably also because expansion and compression does not occur, making the presence of a surfactant reservoir in the watery subphase of the pulmonary gasexchange area unnecessary in bird lungs.

SP-A in Alveolar and Tubular Lungs Although SP-A may not be essential for surfactant function in SP-A knockout mice (31), in vitro studies indicate that SP-A accelerates surfactant lipid adsorption at the air– liquid interface (22). Based on a large body of evidence from both in vivo and in vitro studies, SP-A has been shown to play an important role in innate host defense and regulation of inflammatory processes in the lung (1, 6, 23, 32). Although SP-A immunoreactivity has been detected in the mesobronchi of chicken lungs, which are proximal to the atria with their surfactant-producing type II pneumocytes and the gas-exchanging air capillaries (33), no such SP-A immunoreactivity was found in the atria or air capillaries. However, because polyclonal antimammalian SP-A antibodies were used in this biochemical and immunohistochemical study (33) and the biochemical identity of the reacting protein was not investigated, it remains possible that the observed immunoreactivities identify homologous molecules and not SP-A. Thus, these findings may not provide the final proof of SP-A expression in bird lungs. Such objections are supported by comparative data on SP-A immunoreactivity in various vertebrates. These include immunoblots of chicken lavage, under reducing conditions, where polyclonal anti–SP-A antibodies reacted with bands differing in their molecular weight from mammalian SP-A monomers (34), and findings where SP-A immunoreactivity was absent from the surfactant fraction of both chicken and duck lung lavage (9). The absence of SP-A from the avian type II cell and surfactant, is consistent with the absence of tubular myelin from surfactant of birds (9). A possible explanation for the presence of SP-A in the alveolar lung or of a similar, partially homologous protein in the more proximal airways (mesobronchi) of bird lungs, but its absence from the avian type II pneumocytes and surfactant located in the air capillaries, is based on the essential differences in respiratory physiology between birds and mammals (9, 25, 33). Sedimentation of inhaled particles depends on particle size and inversely correlates with air flow (35). In mammalian lungs, which are aerated via a bellowlike mechanism, airflow is low in small bronchioles and in the alveoli (terminal gas-exchanging structures). On the other hand, in bird lungs, due to sophisticated and pressurecontrolled valve mechanisms, air is forced through the parenchyma (parabronchi, atria, and air capillaries), with high unidirectional flow during both inspiration and expiration


(25, 36, 37) (Figure 1A). Consequently, the bellows-like action of a pulmonary alveolus, which causes low air movement and facilitates precipitation of inhaled particles in this compartment (Figure 1B), is avoided in the gas-exchanging parenchyma of bird lungs and may predominantly take place in other areas (mesobronchi and air sacs) of the bird lungs (2, 25). Support for this theory is provided not only by the location of SP-A (immunoreactivity), but also by the location of macrophages that interact with both inhaled particulate materials and SP-A in mammalian lung alveoli. Although macrophages together with SP-A are present in alveoli, both SP-A and macrophages are absent from the surface of avian air capillaries, as long as these animals live under conditions without significant air pollution (38). Consequently, absence of SP-A from type II pneumocytes and surfactant in avian lung, together with the absence of macrophages in the avian gas-exchange area, as well as the different conditions of gas flow in avian compared with mammalian lungs, point to the possibility of a differential molecular design of surfactant with the potential to function appropriately under the respective biological conditions.

Surfactant Lipid Composition in Alveolar and Tubular Lungs Recent data demonstrate that molecular differences in surfactant composition are also observed in surfactant phospholipids from different vertebrate species or under different developmental conditions. The major molecular species of PC in alveolar surfactant of human adults as well as other mammals and birds is dipalmitoyl-PC (PC16:0/16:0). However, two additional individual PC species, namely palmitoylmyristoyl-PC (PC16:0/14:0) and palmitoylpalmitoleoyl-PC (PC16:0/16:1), which are virtually absent from rigid bird lungs, are selectively secreted into the alveoli of mammalian lungs (10). Interestingly, by contrast with adults, surfactant from more rapidly breathing human infants as well as newborn pigs, rats, and mice contains higher concentrations of PC16:0/14:0 and PC16:0/16:1 at the expense of the main component PC16:0/ 16:0. In adult mammals, PC16:0/14:0 and PC16:0/16:1 concentrations also correlate with respiratory rates (10) (for summary see Table 1). Concentrations of PC16:0/14:0 and PC16:0/16:1 peak at or shortly after term. It is unclear, however, whether biochemical maturation of the surfactant system correlates with morphologic development (cell proliferation and alveolarization) or with specific needs due to exposure to ambient air. In human and porcine lungs the saccular and alveolar periods overlap at term, and alveolarization starts between 80 and 92 d in the pig (term: 115 d) and at 36 wk gestational age in humans. Newborn rat lungs (term: 22 d) are still in their saccular phase at birth, which lasts from 21 d after conception to 4 d after delivery, whereas alveolarization lasts from 4–13 d after delivery (39–41). In guinea-pig lungs (term: 68 d) the saccular phase lasts from 50–60 d after conception, and alveolarization from 60–68 d of gestational age, resulting in alveolar instead of saccular lungs at term (39, 42). Irrespective of these different morphologies at end gestation, concentrations of PC16:0/14:0 are highest around birth in all these mammalian species (10, 43). It is, therefore, hypothesized that increased fractional concentrations of the

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so-called “minor components” PC16:0/14:0 and PC16:0/16:1 are important for maintaining normal lung function (and further development) of mammalian lungs after exposition to ambient air. Surfactant from the rigid bird lung is enriched in PC16:0/ 16:0, but PC16:0/14:0 is virtually absent and PC16:0/16:1 is considerably reduced. Nevertheless, this material displays good adsorption to air–liquid interfaces, which may be due to a sufficient concentration in other liquifying phospholipids such as palmitoyloleoyl-PC (PC16:0/18:1) and SP-B. On the other hand, compressibility of a film of avian surfactant is inferior compared with mammalian surfactant (9). Consequently, one postulated function of PC16:0/14:0 and PC16:0/ 16:1 could be their contribution to dynamic properties of surfactant in the oscillating alveoli. Surfactant from fastbreathing mammals is particularly increased in PC16:0/14:0 and PC16:0/16:1, at the expense of PC16:0/16:0, whereas the major amounts of newly synthesized PC16:0/18:1 in mice, rats, pigs, and humans are retained in the lung tissue rather than secreted into the alveoli (10). Except for PC16:0/16:0, all components possess low gel to sol phase transition temperatures, improving surfactant fluidity at body temperatures around 37⬚C. The need of a surfactant coping with surface dynamics of pulmonary air–liquid interfaces is unquestionably high at respiratory rates of 300–400 strokes per minute (newborn mice and rats) compared with an adult human or pig (12–14 strokes per minute), and the correlation between PC16:0/14:0 and PC16:0/16:1 and respiratory rates implicate roles for these molecules in alveolar surfactant dynamics. However, because improvement of surfactant adsorption can also be achieved by the ubiquitous palmitoyl oleoylPC (PC16:0/18:1) (16) it is possible that PC16:0/14:0 and PC16:0/16:1 either further assist in this process or their functions are yet unknown. One function of the secreted phospholipid surfactant components may be that of regulation and tuning down of immunologic processes in the lungs, particularly in the vulnerable postnatal phase of pulmonary development. Phospholipids, particularly PC, from surfactant are able to inhibit lymphocyte functions, but there is little information on the effect of individual PC molecular species on such processes (44). Moreover, surfactant phospholipids inhibit the respiratory burst of phagocytes, possibly by ameliorating the membrane association of NADPH oxidase components p47phox and p67phox. Although other isolated surfactant PC species were not investigated, the effect of PC16:0/16:0 together with phosphatidylglycerol was inferior to native surfactant, and even higher concentrations of pure PC16:0/16:0 showed no suppressing effects (45, 46). By contrast, preliminary data have demonstrated that PC16:0/16:1 and PC species with a short saturated fatty acid in the sn-2 position of the PC molecule (such as PC16:0/14:0) are more potent in inhibiting radical formation than other components in vitro (47). Such inhibition of the respiratory burst may be even more important in the presence of SP-A in the developing alveolus, where SP-A acts as an opsonin facilitating uptake of microorganisms via binding to surface receptors on macrophages, than in avian pulmonary air capillaries that are devoid of SP-A and macrophages (9, 38) (see above). Such information, together with the finding

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TABLE 1

Comparison of surfactants from different locations and animals compared to alveolar surfactant from large mammals with lower respiratory rates

Surface tension function under dynamic conditions Tubular myelin SP-A SP-B SP-C PC16:0/16:0 PC16:0/14:0 PC16:0/16:1

Airway surfactant from mammalian lungs

Surfactant from the rigid tubular bird lung

Alveolar surfactant from mammals with high respiratory rates

Decreased adsorption Increased minimal surface tension Absent Decreased Absent/decreased Absent/decreased Unchanged (55%*) Unchanged (about 10%*) Unchanged (about 10%*)

Good adsorption Increased minimal surface tension Absent Absent Present Absent Increased (70%*) Absent (⬍ 2%*) Decreased (5%*)

Rapidly reaches minimal surface tension near zero Present Present Present Present Decreased (down to 40%*) Increased (up to 25%*) Increased (up to 22%*)

Surfactant from airways, bird lungs, and alveolar surfactant from mammals with high respiratory rates (up to 400 breaths/min, as in 1-wk-old mice) are compared to alveolar surfactant from large mammals with lower respiratory rates (12–14 breaths/min as in the human and pig). Data are summarized from Refs. 8–10, where identical techniques for functional and biochemical analyses have been used. *Percent of total phosphatidylcholine (PC).

that in airway surfactant from mammals, PC16:0/14:0 and PC16:0/16:1 are in the same range as in their alveolar surfactant, supports the view that, in addition to their effects on spreading to the air–liquid interface, the latter molecules may be particularly important for the regulation of inflammatory processes in the pulmonary periphery. Conclusion The data reviewed here indicate that the molecular design of lung surfactant may be adapted to morphologic and physiologic characteristics of the respective organs and structures, whether alveolar or tubular. Such differences include surfactant proteins as well as phospholipid composition. Future studies will have to explore in more detail the relation between pulmonary structure and physiology and surfactant composition. Such investigations may improve our concepts for the future application of surfactant or its components under different clinical conditions and for different purposes, for example to supplement surface-active, antimicrobial, or immunoregulatory functions, and according to whether targeted at the conducting airways or alveoli. Acknowledgments: This study was supported by NIH R37 HL34788, DFG Ha1959/2, and The Royal Brompton and Harefield Charitable Fund No B0437.

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