PERSPECTIVE
Surfactant Protein B Deficiency Worsens Hyperoxic Injury to the Alveolar Epithelium

Judy Hickman-Davis and Sadis Matalon

Departments of Anesthesiology and Comparative Medicine, University of Alabama at Birmingham, Birmingham, Alabama

The epithelial lining fluid of normal lungs contains pulmonary surfactant synthesized by alveolar type II cells. This lipoprotein complex consists mainly of phospholipids and at least four different associated proteins, labeled surfactant proteins A, B, C, and D (SP-A, SP-B, SP-C, and SP-D). The main functions of surfactant are to lower the surface tension of the air-liquid interface and stabilize alveoli at low lung volumes. These functions decrease the work of breathing and prevent extravasation of fluid into the alveolar space.

All surfactant apoproteins are synthesized in alveolar type II (ATII) and Clara cells, except for SP-C, which is synthesized in ATII cells only. The hydrophobic surfactant proteins SP-B and SP-C enhance adsorption of surfactant lipids to the air-liquid interface and decrease the surface tension of the monolayer during area compression (1). SP-B belongs to the family of saposin-like proteins along with natural killer (NK)-lysin and amoebopore (2). Saposins are proteins that activate lysosomal hydrolases and have antibacterial properties. SP-B is essential for surfactant function, for homozygous genetic deficiency of this protein is lethal to both humans and mice and is associated with abnormal processing of SP-C (3, 4). SP-A and SP-D belong to the superfamily of collagenous lectins known as collectins and are thought to participate in the regulation of surfactant homeostasis, tubular myelin formation, and nonspecific innate immune responses of the lung (5). In addition, SP-A cooperates with SP-B and SP-C in lowering surface tension of surfactant monolayers, especially in pathologic situations (6). SP-A-deficient mice (SP-A^/) have normal surfactant function but decreased ability to clear bacteria from their lung spaces (7, 8).

Several experimental studies have provided evidence for oxidant-mediated injury to the lung under clinically relevant conditions. Prolonged exposure of animals to high concentrations of oxygen (> 95% O₂) damages the alveolar epithelium and the pulmonary surfactant, leading to the development of permeability-type alveolar edema, arterial hypoxemia, and death. For example, rabbits exposed to 100% oxygen for 72 h display decreased phospholipid levels, increased levels of albumin and surface tension in the bronchoalveolar lavage fluid (BALF), and decreased lung compliance (C) and total lung capacity (TLC) (9). ATII cells, isolated from the lungs of these rabbits, exhibited a decreased ability to synthesize surfactant lipids. Exposure of hamsters to 100% oxygen resulted in decreased phosphotidylglycerol content of the total phospholipid, as well as decreased SP-A, SP-B, and SP-C messenger RNA (mRNA) (10). Patients with fulminate acute respiratory distress syndrome (ARDS) or at risk for ARDS have decreased total alveolar phospholipid and SP-A and SP-B levels. Furthermore, when tested with an oscillating bubble surfactometer, their surfactant exhibited abnormally high minimum surface tension upon dynamic compression (11). On the basis of the aforementioned studies, it was concluded that excessive amounts of reactive oxygen and nitrogen species damaged critical components of the pulmonary surfactant system. The next logical step was to identify whether animals with decreased levels of surfactant apoproteins exhibit higher susceptibility to oxidant injury.

In this issue, Tokieda and coworkers at the University of Cincinnati utilized gene-targeted mice (SP-B^+/) in an attempt to identify the effect of decreased levels of SP-B on hyperoxia-induced lung injury in vivo (12). Heterozygous SP-B gene-targeted mice produce ~ 50% of the SP-B mRNA and protein of normal SP-B^+/+ mice and provide an excellent example of gene dosage for control of gene expression (13).

As shown in Figure 1 in the article beginning on page 463 of this issue, SP-B^+/ mice have slightly decreased specific C and normal TLC while breathing air. However, after breathing 95% oxygen for three days, the mice had decreased C and TLC and markedly increased alveolar permeability to albumin as compared with SP-B^+/+ mice (Figures 1 and 4). Based on these data, one may conclude that injury to SP-B by reactive oxygen and nitrogen species may further depress already low levels of this apoprotein and result in abnormal surfactant function. Indeed, previous studies indicate that SP-B/SP-C mixtures exposed to reactive nitrogen species do not decrease the minimum surface tension of surfactant phospholipids in vitro (6). It should be pointed out that these data provide strong evidence that normal surfactant function is crucial for the maintenance of low permeability to solute. These data also agree with previous reports suggesting that surfactant replacement reverses the hyperoxia-induced increased alveolar permeability to solute (9, 14).

Interestingly, data shown in Figures 2 and 3 indicate that exposure to hyperoxia results in significant increases in both SP-B mRNA and SP-B protein levels in the BALF of both SP-B^+/ and SP-B^+/+ mice. These data are consistent with previous reports from this group indicating that hyperoxia increases levels of all surfactant apoproteins in rats (15). At first glance, these data appear contradictory with the physiologic findings: although, during exposure to hyperoxia, SP-B levels in SP-B^+/ mice were about 50% of that seen in the SP-B ^+/+ mice, they were still much higher than levels found in normal air-breathing mice of either genotype. What then can account for the decreased C and TLC of these mice? In other words, how did the SP-B^+/ mice develop surfactant deficiency in the presence of higher than normal levels of hydrophobic surfactant apoproteins in their BALF?

There are several explanations that may account for these findings. First, as shown in Figure 3, the much higher levels of albumin in the BALF of SP-B^/ mice may have interfered with the ability of surfactant to reach a minimum surface tension. Second, as pointed out by the authors, SP-B present in the BALF of SP-B^/ mice may have been sloughed from broken cells and may be nonfunctional. Indeed, in contrast with the increase in SP-B in BALF, cytoplasmic staining for SP-B, proSP-B, and SP-C was decreased within the lungs of both genotypes, with a complete lack of staining for SP-B and proSP-B in the ATII cells of SP-B^+/ mice. The association of proSP-B primarily with sloughed cells and cellular debris within the airways may partly explain the measured increase in SP-B levels in the BALF. Measurements of surface tension of the BALF of these mice and reconstitution of SP-B with surfactant lipids may have provided additional insight as to the mechanisms involved. The study by Tokieda and coworkers highlights the importance of correlating morphologic and biochemical changes with functional indices of surfactant function. Moreover, data shown in Table 2 clearly point out that hyperoxia damages ATII cells of both SP-B^+/ and SP-B^+/+ mice and decreases their ability to produce SP-B. Thus, previous reports of increased levels of surfactant apoproteins in the BALF of mice exposed to lethal hyperoxia should be interpreted with caution. It should be stressed that exposure to sublethal levels of hyperoxia may increase both the number of ATII cells and their ability to produce surfactant (16).

The discovery of SP-B and the importance of this protein to surfactant replacement formulations provide an incentive to better understand the gene regulation, biology, and function of this protein. Tokieda and associates speculate that exogenous SP-B may be a useful therapeutic strategy for various lung disorder in patients with mutant SP-B alleles. However, infants homozygous for the SP-B deletion are unresponsive to replacement therapy. These observations emphasize the need for further investigation and the development of expression systems and transgenic models, which would be critical for the understanding of the interplay between the surfactant proteins and phospholipids in vivo.

	Footnotes

Abbreviations: alveolar type II cells, ATII; acute repsiratory distress syndrome, ARDS; bronchoalveolar lavage fluid, BALF; lung compliance, C; surfactant protein, SP; total lung capacity, TLC.

(Received in original form August 10, 1999).

Acknowledgments: The authors are currently supported by grants RR00149 (J.H.D.), HL31197, and HL51173 (S.M.) from the National Institutes of Health.

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