Alveolar lining fluid normally contains proteins of important physiological, antioxidant, and mucosal defense functions [such as albumin, immunoglobulin G (IgG), secretory IgA, transferrin, and ceruloplasmin]. Because concentrations of plasma proteins in alveolar fluid can increase in injured lungs (such as with permeability edema and inflammation), understanding how alveolar epithelium handles protein transport is needed to develop therapeutic measures to restore alveolar homeostasis. This review provides an update on recent findings on protein transport across the alveolar epithelial barrier. The use of primary cultured rat alveolar epithelial cell monolayers (that exhibit phenotypic and morphological traits of in vivo alveolar epithelial type I cells) has shown that albumin and IgG are absorbed via saturable processes at rates greater than those predicted by passive diffusional mechanisms. In contrast, secretory component, the extracellular portion of the polymeric immunoglobulin receptor, is secreted into alveolar fluid. Transcytosis involving caveolae and clathrin-coated pits is likely the main route of alveolar epithelial protein transport, although relative contributions of these internalization steps to overall protein handling of alveolar epithelium remain to be determined. The specific pathways and regulatory mechanisms responsible for translocation of proteins across lung alveolar epithelium and regulation of the cognate receptors (e.g., 60-kDa albumin binding protein and IgG binding FcRn) expressed in alveolar epithelium need to be elucidated.
- alveolar epithelial cells
- secretory component
- immunoglobulin G
macromolecule transport across alveolar epithelium has been investigated over the years, but little is known to date concerning the mechanisms and underlying pathways regulating transalveolar protein clearance. Alveolar protein clearance is fundamental to the resolution of the transudate and exudate after hydrostatic- and especially high permeability-type pulmonary edema. The inability to clear excess proteins from air spaces is associated with poor prognosis in patients with alveolar flooding pulmonary edema. Clearance of edema fluid via active ion transport processes raises the oncotic pressure that, in the absence of efficient clearance mechanisms for proteins, slows alveolar fluid clearance. Patients with acute respiratory distress syndrome (ARDS), for example, have large quantities of precipitated protein in their air spaces (3), and nonsurvivors have three times more protein in their air spaces than survivors (21). In addition, excess serum proteins in alveolar edema fluid may lead to precipitation and modification of proteins (e.g., by reactive oxygen and nitrogen species). Although macrophages may contribute to clearance of the excess proteins, the integrity of the alveolar epithelial barrier may not be readily restored and protein clearance mechanisms may be severely compromised. For example, cationic amino acids and peptides that result from degraded and metabolized serum proteins in the alveolar air spaces are known to increase the leakiness of solutes into alveolar fluid by disruption of tight junctional pathways. Excess proteins in the air spaces may also promote progression of end-stage fibrosis in ARDS since growth of fibrous tissues requires hyaline membranes (67, 116). Moreover, protein accumulation in alveoli may reduce the surface activity of surfactant, further contributing to collapse of alveoli and alveolar flooding (56).
The topic of protein clearance in the lung (especially across the alveolar epithelial barrier that covers almost all air space surfaces) is physiologically important and clinically relevant. Because concentration of serum proteins in alveolar fluid increases in injured lungs (such as in congestive heart failure, high-permeability edema, and inflammation), understanding how the alveolar epithelium handles protein transport is necessary for developing better therapeutic approaches to restore transalveolar clearance of fluid. Moreover, mechanistic information on how exogenous and endogenous proteins traverse the air-blood barriers of the lung is likely to provide new insights into improved strategies for pulmonary delivery of exogenous protein drugs into the systemic circulation and targeting drugs to lung parenchymal cells via specialized processes (e.g., receptor-mediated uptake).
This review deals with protein transport across the respiratory epithelial (mostly alveolar) barrier. As review articles and book chapters on the subject of lung protein clearance and alveolar epithelial protein transport have appeared recently (41,53), we have attempted here to summarize the field, provide mechanistic insights, and evaluate the most significant findings of recent years.
PROTEIN TRANSPORT ACROSS LUNG EPITHELIAL BARRIERS
Studies Using Whole Lung Preparations
The lung comprises ∼40 different types of cells. Air spaces of the lung can be categorized as the air-conducting region and the gas-exchanging region. Up to terminal bronchioles, the air spaces are lined with airway epithelial cells that are different in morphology and function from those lining the distal portion of the lung. The distal air spaces of the lung are lined with a continuous epithelium comprising two major types of alveolar epithelial cells (type I and type II pneumocytes) joined by tight junctions (zonulae occludentes). This structural arrangement is consistent with the fact that pulmonary epithelium is the primary barrier to solutes compared with the pulmonary capillary endothelium (112). The airway and alveolar epithelial barriers offer significant resistance to the diffusion of electrolytes and small hydrophilic solutes compared with endothelial barrier lining the lung vasculature (112). Tightness of epithelial barriers as regulated by zonulae occludentes helps to keep the air space relatively dry for efficient gas exchange.
Lung Protein Transport Studies Using Intact Lung Models
This section is divided into two major areas dealing with lung protein clearance in nonpathological and pathological states. Because the transalveolar pathways involved in protein clearance are poorly understood, the term “lung protein clearance” (instead of “alveolar protein transport”) is chosen to stress this point. Similarly, “distal air spaces” rather than “alveolar air space” is used throughout the sections on intact lung studies.
Protein Clearance Studies Using Intact Lung Models Under Nonpathological Conditions
Rate of protein clearance is slower than fluid clearance from distal air spaces under normal conditions.
Protein clearance rate via respiratory epithelial tract lining the distal air spaces of the lung is ∼1–2%/h for albumin when normal lungs were instilled intratracheally with this serum protein (41, 53). The lung clearance rate for albumin appears to be similar over many animal species, including human (6, 7, 52,96). The rate of elimination of intratracheally instilled125I-albumin from the distal air spaces of lungs of awake sheep occurred at a monoexponential rate over the course of 6 days (6). In striking contrast, intratracheally instilled fluid cleared from the air spaces at a rate of 10–25%/h with some variance among different species (7, 8, 105). Because instilled fluid clears faster than proteins, oncotic pressure in edema fluid tends to rise over time and may subsequently contribute to a decrease in lung fluid clearance (72, 77, 78, 105). These reports suggest that protein clearance from edema fluid can become a limiting step in the resolution phase of alveolar pulmonary edema.
Respiratory epithelial tract lining distal air spaces is the limiting barrier for protein clearance.
The relative resistance of the alveolar epithelium vs. capillary endothelium to the passage of protein has been studied using morphological and physiological techniques. Ultrastructural protein tracers (e.g., horseradish peroxidase and cytochrome c) generally do not cross the epithelial junctions but are found in the interstitial spaces when perfused in the vasculature (except for catalase and ferritin, which do not the penetrate endothelial junctions). An increase in protein permeability is seen at high alveolar distending pressures (47, 87, 97-99, 123), suggesting that the integrity of the air-blood barrier is needed to prevent the abnormal leak of plasma proteins into the epithelial lining fluid of distal air spaces. On the basis of reflection coefficients for small solutes, Taylor and Gaar (112) estimated the radii for equivalent water-filled pores to be 0.5–0.9 nm in respiratory epithelial tract and 6.5–7.5 nm in capillary endothelium. The epithelium lining the distal air spaces of the lung was responsible for 92% of the resistance to 125I-albumin flux across the air-blood barrier of adult sheep lung with chronic lung lymph fistulas (50). These reports are consistent with the generally accepted concept that the respiratory epithelial barrier lining distal air spaces, rather than the interstitium and endothelium, is the principal and limiting barrier for lung protein clearance.
Lung protein clearance is dependent on molecular size.
The rate of protein absorption from the distal air spaces appears to be dependent, in part, on molecular size. In this regard, Effros and Mason (35), upon analysis of various published data on lung epithelial protein permeability, showed an apparent inverse (albeit not strictly reciprocal) relationship between molecular weight and rate of lung clearance of molecules of different sizes. A later study on alveolar epithelial permeabilities of urea, sucrose, inulin, and dextran (60–90-kDa range) in saline-filled dog lungs also showed a similar relation between the size of molecules and permeabilities (113). In support of these findings, a larger permeability-surface area product for sucrose than for albumin in rabbit lungs was reported (120), and the bioavailability of intratracheally instilled proteins exhibited lower values for larger proteins (42, 43). Analysis of the amount of radiolabeled proteins that remained in the human lung 8 h after intratracheal instillation indicated that larger proteins cleared at a slower rate than smaller proteins (52). Moreover, the fraction of total protein as albumin in edema fluid was initially greater than in plasma, although after 10 h the albumin fraction significantly decreased from 0.62 to 0.58 in 10 patients with resolving pulmonary edema, presumably due to faster clearance of albumin from the air spaces compared with slower clearance of larger proteins (e.g., immunoglobulins). The normal air-blood barrier of human lungs appears to differentially restrict the passage of large proteins (e.g., IgM, fibrinogen, and catalase) as evident by the finding that concentrations of these macromolecules in bronchoalveolar lavage (BAL) fluid were extremely low compared with the smaller proteins (e.g., albumin) (55).
Lung protein clearance may involve catabolic pathways.
Catabolism of proteins in the airways followed by absorption of smaller, degraded protein fragments was first proposed by Drinker and Hardenbergh (33), although it does not appear to play a predominant role in protein clearance in normal lungs. In general, >95% of the protein tracers (such as albumin) instilled into the distal air spaces may reach the circulation intact (5, 6,42-44, 48, 77). After intratracheal instillation of radioiodinated albumin in sheep lungs, trichloroacetic acid-soluble radioiodine over 6 days in lung lavage, urine, thyroid, and feces all increased significantly after 2 days, but >80% of the extrapulmonary radioiodine was still associated with the parent molecule (6). In contrast, much greater protein degradation was noted in studies in perfused dog (101) and rabbit (15) lungs. Whether greater protein degradation may have occurred from proteolysis in the bronchial epithelium (62) is unknown, although another group using the dog lung model and similar techniques found that 95% of the tracer protein was cleared into the perfusate as intact molecules (48). These reports underscore the difficulty inherent in intact lung studies and highlight the need for caution in interpretation of transport data from intact lung models. For smaller proteins and peptides, variable amounts of intact calcitonin, insulin, and desaminoarginine vasopressin (DDAVP) have been reported to be cleared from the air spaces (24, 42,52). Degradation by peptidases localized at apical surfaces of respiratory epithelial cells may be the predominant mechanism for clearing most peptides (e.g., vasoactive intestinal peptide) (4) and some smaller proteins [e.g., gastrin, 2.2 kDa, (R. Hastings, personal communication)] from the lung air spaces, although endocytosis followed by lysosomal proteolysis in the epithelium and endothelium cannot completely be ruled out from these studies.
Summary of lung protein clearance in nonpathological states.
Lung protein clearance in normal mammalian lungs appears to be relatively slower than liquid clearance, and thus it could be a “bottleneck” in the resolution phase of edema fluid secondary to rising concentration of proteins. Alveolar clearance of proteins appears to be size dependent because the larger proteins, in general, clear at a slower rate than the smaller ones. Some proteins (i.e., albumin) may be cleared relatively intact, whereas smaller proteins and peptides may undergo significant degradation. The mechanisms and specific pathways underlying lung protein clearance remain largely unknown, necessitating studies in simpler models such as primary cultured respiratory epithelial cells.
Lung Protein Clearance Under Pathological Conditions
Protein clearance from the air spaces has been studied in lungs subjected to pathological conditions including injury and inflammation, immune response, and alveolar proteinosis. Of note is the general observation that pulmonary edema fluid contains a high concentration of protein, often approaching 65% of the concentration in serum in lungs subjected to hydrostatic edema, and even higher in permeability edema (18, 40, 52, 79, 106). The mechanism of lung protein clearance in the face of such high protein concentration in the air space fluid is not fully understood. Here, we review studies of protein clearance in lungs under pathological conditions.
Protein clearance increases in lungs injured by infectious and inflammatory agents.
Alveolar epithelium is remarkably resilient, as seen by the ability of the epithelium to continue serving as a barrier to fluid flow and solute flux in the absence of pulmonary blood flow or ventilation for several hours (59, 96). The respiratory epithelial tract lining the distal air spaces of the lung is relatively resistant to injury by infectious agents (89, 90). Instillation of 1010 live Pseudomonas aeruginosa into the distal air spaces of sheep lung is required to double the rate of movement of labeled albumin over 24 h across the epithelium in either the air space-to-interstitial or opposite direction (88). Of particular note in the latter study is that intratracheal instillation of IgG antibody to P. aeruginosa prevented the increase in respiratory epithelial permeability, whereas systemic perfusion of the same antibody protected the endothelial barrier, suggesting that the properties of the epithelial and endothelial barriers are independently regulated. Although lung airways (and type II cells) secrete defensins as a critical component of the host defense response against infectious organisms (1, 34, 60), the exact role of defensins in mediating alveolar epithelial permeability alterations is unknown.
Pulmonary inflammation induced by intratracheal instillation of Escherichia coli endotoxin or ferritin in rat lungs caused dose-dependent increases in the lung clearance of bovine IgG (bIgG), BSA, and DDAVP such that the changes in lung clearance of these markers were greater for bIgG and BSA than for DDAVP (43). With severe inflammation following the use of large concentrations of ferritin, bIgG clearance approached the clearance of the two smaller tracers (43). These reports are consistent with the hypothesis that clearance of BSA and bIgG via the paracellular pathways of the respiratory epithelial barrier is modest in normal lungs, but that inflammation causes disruption of paracellular routes to induce significant leak of these two larger proteins. Unfortunately, the possible stimulatory effects of inflammatory agents on transcellular processes (i.e., transcytosis) involving the translocation of these proteins were not addressed, underscoring the need for further studies in the whole lung model. In addition, damage to air-blood barrier function in noncardiogenic edema, but not in cardiogenic edema, may be responsible for the faster appearance of intravenously injected 131I-albumin into BAL fluid of patients with ARDS compared with patients with congestive heart failure (46). These BAL data suggest that some serum proteins are present in measurable quantities in normal alveolar fluid and that alveolar fluid concentrations of such proteins are affected, in part, by alveolar epithelial transport processes including paracellular leak, active ion transport, and transcytosis.
Persistent protein clearance in lungs subjected to infectious and inflammatory agents requires intact alveolar epithelium.
Damage to the epithelial barrier effectively prevents the clearance of excess liquid and proteins from the air spaces. Resolution of pulmonary edema requires a functional and intact alveolar epithelial barrier. The respiratory epithelium lining the distal air spaces appears to have the capacity to recover rapidly from injury and thus to restore the fluid clearance mechanism (79). Studies showed that the net clearance of alveolar liquid begins as early as 4 h after oleic acid-induced permeability edema in sheep lungs (122). Patients with high-permeability pulmonary edema were also reported to begin clearing edema within 12 h of onset and exhibited progressive concentration of edema fluid proteins in parallel with decrease in edema on the chest radiograph (79). Lung injury appears to cause elevation of BAL concentrations of the larger molecules and antioxidant proteins (i.e., catalase, ceruloplasmin, and transferrin) (40, 55, 106). Interestingly, the larger proteins (catalase, IgM) remain in the alveolar fluid even after the concentrations of the smaller molecules, such as albumin, returned to normal levels (55). The exact mechanisms for this exclusion phenomenon observed during injury and repair of the lung are unclear.
Increased neutrophil trafficking fails to increase lung protein clearance, but free radicals generated by inflammatory cells promote clearance.
Influx of neutrophils into air spaces occurs with lung inflammation, but it does not increase in protein permeability across the respiratory epithelial tract. For example, intratracheal instillation of leukotriene B4 caused a large increase in neutrophil numbers in BAL fluid, whereas the total protein content of lung lavage from healthy human volunteers was not changed (73). Similarly, intratracheal instillation of a low dose of E. coli endotoxin resulted in neutrophil recruitment into air spaces of sheep lungs without a significant change in fluxes of labeled protein across the air-blood barrier in either air space-to-vascular or opposite direction (121). After challenge of lungs with large amounts of inflammatory agents, lung protein permeability increased secondary to generation of free radicals and other reactive metabolites released by inflammatory cells, but not by the recruited inflammatory cells per se (43). Intratracheal instillation of ferritin resulted in neutrophil recruitment with a large increase in lung protein clearance, whereas coadministration with a hydroxyl radical scavenger, mannitol, abrogated the increase in passage of protein markers into vascular space from the air spaces (43). Thus this response is most likely the result of radicals formed by the iron-catalyzed reaction rather than the recruited neutrophils. It remains to be determined whether oxidant stress can increase transcellular transport of proteins across air-blood barrier in these experiments, and if so, by what mechanism.
Summary of lung protein clearance in pathological conditions.
Changes in barrier function (especially paracellular leak) of the respiratory epithelial tract have a major impact on lung protein clearance in lung injury, whereas activation of transcellular processes in the respiratory epithelial barrier may be important in immune responses. In injured lungs, the degree of intactness of the respiratory epithelial barriers appears to be a prerequisite for persistent protein clearance.
Lung Protein Clearance Is Modulated by Soluble Factors
Recent studies in granulocyte/macrophage colony-stimulating factor (GM-CSF) knockout mice have suggested that defective macrophage function leads to alveolar proteinosis, a rare disease characterized by accumulation of large amounts of surfactant apoproteins and other proteins in alveoli (56). Mice lacking GM-CSF developed pulmonary disease marked by progressive accumulation of surfactant apoproteins and lipids in the alveolar air space (31, 32), hallmarks of alveolar proteinosis. In these animals, surfactant apoprotein mRNA levels and synthesis were normal, but clearance of surfactant protein A (SP-A) was decreased, suggesting the impairment of surfactant apoprotein recycling pathways. The alveolar air space contained enlarged, foamy macrophages laden with lipids that may have resulted from a defect in surfactant clearance or catabolism in these macrophages. Selective overexpression of GM-CSF in type II cells in mice (57) abrogated the abnormalities of surfactant homeostasis and normalized SP-A, SP-B, and phospholipid levels without changes in mRNA levels of SP-A, SP-B, or SP-C. These data indicate that GM-CSF regulates the clearance and catabolism of surfactant apoproteins and that endocytosis and phagocytosis by alveolar macrophages are essential for these processes.
Pathways and Mechanisms of Lung Protein Clearance
The normal lung lavage fluid contains serum proteins ranging from albumin to polymeric immunoglobulins at concentrations much lower than those in blood. However, the pathways and mechanisms responsible for protein transport in the lung remain generally unclear. Albumin is normally found in lavage fluid at a concentration of ∼8–10% of that in blood, whereas the concentration of serum proteins of 100 kDa in lavage fluid is 1% of their concentrations in blood and that of 10 kDa proteins is ∼20–25% of that found in blood. Thus there is an inverse relationship between the molecular masses of serum proteins and their concentrations in lavage fluid. In this regard, the leak of serum proteins from blood and interstitial spaces into lung air spaces may occur via restricted passive diffusional routes rather than through nonspecific leak pathways that allow equal rates of transport for small and large molecules. As for the participation of respiratory epithelial cells in transport of proteins, the type I pneumocyte, with a much greater overall surface area in the lung, is likely to be important in transcytosis of proteins and other macromolecules presented to the air spaces (70). Perivascular and peribronchiolar cuffs known to be formed in the bronchoalveolar region after instillation of fluid in the air spaces or a sudden rise in left atrial pressure may be the leakage sites for plasma protein in either direction (45, 107,108). There is also the possibility of a receptor-mediated (and perhaps adsorptive) endocytosis in the translocation of serum proteins and other related substances (e.g., surfactant proteins) (61, 66,123-125). Maclura pomifera agglutinin and cationic ferritin bind to the surface of alveolar type II pneumocytes, and these markers are also found to decorate coated and uncoated pits in the plasmalemma as well as intracellular vesicles (123,124). Subfractions of a natural surfactant containing proteins with molecular masses of 26–36 kDa were preferentially taken up by intact rat lungs to a greater extent than fractions lacking these apoproteins on intratracheal instillation of the macromolecules (125). The specific endocytic mechanisms for this enhanced uptake of surfactant fractions containing low-molecular-mass proteins are not clearly defined.
Autologous IgG is found in alveolar epithelial cell coats, tubular myelin, and free granular deposits, whereas autologous albumin is present on the surfaces of type II pneumocytes and in vesicles located on type I cells (9, 10). Other investigators showed that type I and type II cells take up ferritin and horseradish peroxidase into vesicles (47, 98, 123), whereas type II cells internalize SP-A from the alveolar space by receptor-mediated endocytosis (94). Both type I and type II cells have been shown to phagocytose foreign particles (22, 54, 63). Finally, clathrin-coated pits are present in alveolar type I and type II cells, whereas caveolin-coated plasmalemmal vesicles (i.e., caveolae) are present in both pulmonary vessel endothelial cells and alveolar type I epithelial cells, suggesting possible transcytosis of proteins via these structural features (16, 64, 85). Type II pneumocytes may not express caveolin (16), although recent studies using more specific reagents indicate otherwise (61). Various studies have invoked the important role of transcytosis in alveolar epithelial cells.
Internalization of exogenous proteins occurs via endocytosis, a process involving invagination of a small region of the plasma membrane to form new intracellular membrane-limited vesicles (∼50–100 nm in diameter). Most eukaryotic cells continually engage in endocytosis that can be categorized as pinocytosis (fluid-phase endocytosis or nonspecific endocytosis), adsorptive endocytosis, and receptor-mediated endocytosis (84). In pinocytosis, endocytic vesicles nonspecifically internalize small droplets of extracellular fluid (and thus any material dissolved therein). Pinocytic uptake does not saturate with the concentration of exogenous proteins and is not competed by the presence of other macromolecules. Pinocytosed proteins may undergo sorting and subsequent delivery to contralateral cell membranes (i.e., via transcytosis). For example, a fluid-phase transport marker, horseradish peroxidase, is transcytosed across alveolar epithelium, albeit with a much lower rate than that estimated for albumin or IgG (61, 75, 76). In adsorptive endocytosis, exogenous proteins are adsorbed to the cell membrane or glycocalyx by electrostatic interaction before invagination of endocytic vesicles, making the available cell membrane surface area the limiting factor responsible for the uptake. Uptake via adsorptive endocytosis thus saturates at very high concentrations of exogenous proteins due to the limited availability of adsorption sites. Some competition may occur for the adsorption site(s) if the interaction between one macromolecule and the site is displaceable by other macromolecules. In receptor-mediated endocytosis, a specific receptor (or binding site or binding protein) on the cell surface binds relatively tightly to the exogenous protein (i.e., the ligand) that it recognizes, followed by invagination of the region of cell plasma membrane where the receptor-ligand complex resides. Receptor-mediated endocytosis can occur by at least two different vesicle systems, one coated with clathrin and the other with caveolin. Endocytic vesicles that bud from cell membrane become the transport vesicles involved in the sorting of ligand-receptor complex or dissociated ligands and receptors and the delivery of these cargoes to various targets, including contralateral cell membrane (thus completing transcytosis). Information on transcytosis of proteins in the apical-to-basolateral direction is rather limited. Recent reviews provide a good summary of current information on endocytosis (2, 83, 84).
Studies of Protein Transport Using In Vitro Respiratory Epithelial Models
As explained earlier, lung-specific mechanisms for protein transport are not clearly defined. On the basis of studies of protein transport in nonrespiratory tissue, the general consensus is that proteins predominantly traverse normal epithelial as well as endothelial barriers via endocytotic processes, including fluid-phase (i.e., nonspecific pinocytosis), receptor-mediated, and adsorptive endocytosis (limited by available cell plasma membrane area that invaginates to form vesicles and exhibits saturation at supraphysiological concentrations). In injured epithelium, the leak of proteins via paracellular pathways may become the predominant pathway, whereas the injurious agent may or may not stimulate transcytosis per se.
Specific information on pathways and mechanisms for alveolar epithelial protein transport has been elusive thus far due, in part, to the complex anatomical arrangement of the mammalian lung. The preponderance of existing transport data on numerous solutes (e.g., small hydrophilic solutes including ions, amino acids, sugars, peptides, and proteins) were obtained utilizing the whole lung approach. Dissection of data pertinent to the alveolar epithelial barrier from such a complex experimental model is a formidable task because the model inherently possesses various unknowns. These include solute distribution profiles, surface areas available for transport, and unstirred layers. Moreover, multiple biological barriers are arranged in series (epithelium, interstitium, and endothelium) as well as in parallel (airway and alveolar epithelia). Thus separation of the potential barriers and study of each barrier in isolation become essential for gaining insights into the specific mechanisms of transport of a given solute across the individual barrier such as the alveolar epithelium.
An in vitro model of primary cultured cell monolayers grown on permeable supports (20) has played a pivotal role in the mechanistic investigation of vectorial transport of ions and solutes across the mammalian alveolar epithelial barrier. In brief, freshly isolated and purified rat type II alveolar epithelial cells, when cultivated on tissue culture-treated Nuclepore filter membranes, form confluent cell monolayers starting from ∼3 days of culture. These primary cultured rat alveolar epithelial cells-on-filters exhibit barrier properties of “tightness” against leak of both ions and fluid, along with active ion absorption properties replicating in vivo observations in lungs. The tight rat alveolar epithelial cell monolayers exhibited morphological features of both protuberant nuclei and thin cytoplasmic extensions (which resemble those found in type I pneumocytes in vivo) from day 3 onward (19), indicating that type II cells in culture gradually acquire morphological traits of type I cells. In parallel with these morphological changes, apically localized epitope(s) showed reactivity with rat type I cell-specific monoclonal antibodies from day 3 onward (12, 13, 23). The morphological features and alveolar type I cell-specific antibody reactivity in these primary cultured rat pneumocytes grown on permeable supports appeared to be preserved for up to 60 days in culture (K.-J. Kim and E. D. Crandall, unpublished data). It is now widely accepted that type II cells differentiate into type I (-like) cells in primary culture as revealed by changes in morphological appearance (19), lectin binding properties (28), reactivity with type I cell-specific monoclonal antibodies (23, 29), and other biochemical (e.g., progressive loss of surfactant protein expression) and molecular [e.g., progressive expression of T1α, water channel type 5 (aquaporin-5), and caveolin-1] markers (13, 16, 85,92).
It is important to note that ∼98% of the total surface area in normal mammalian lungs in vivo is covered by alveolar epithelial cells. The distal air spaces are covered largely by type I cells (>95% of total air space of distal lung). In this regard, the tight alveolar epithelial cell monolayer may be a very useful model for the investigation of in vivo properties of the alveolar epithelial barrier. The rat pneumocyte-on-filter model exhibited spontaneous potential difference >10 mV (apical side negative), resistance >2,000 Ωcm2, and short-circuit current of ∼5 μA/cm2 (20, 65). This widely used rat type I cell-like monolayer represents a reliable functional barrier for the study of transport-related issues of alveolar epithelium.
Next, we present mechanistic information for alveolar epithelial transcytosis and metabolism of various exogenous proteins (i.e., ovalbumin and horseradish peroxidase) and dextrans as well as several endogenous proteins [i.e., albumin, transferrin, IgG, and polymeric immunoglobulin A (pIgA)] found in alveolar lining fluid of normal mammalian lungs.
Transport of Horseradish Peroxidase, a Fluid-Phase Endocytosis Marker, and Dextrans Across Alveolar Epithelial Cell Monolayers
To begin delineation of the mechanisms of alveolar epithelial macromolecule transport, studies have addressed the transport properties of alveolar epithelial cell monolayers using several tracers, including horseradish peroxidase (which is widely used as a pinocytosis or fluid-phase marker). With the use of tight rat pneumocyte monolayers and enzymatic assay for horseradish peroxidase, the apparent permeability coefficient (P app) of alveolar epithelium for horseradish peroxidase (∼40 kDa) was determined to be 7 × 10−9 cm/s (75). Horseradish peroxidase transport across alveolar epithelial barrier was neither concentration nor direction dependent (75). Lowering temperature from 35°C to 4°C decreasedP app for horseradish peroxidase by ∼80%, suggesting that horseradish peroxidase traversed primarily via nondiffusional routes across the alveolar epithelium. Interestingly,P app for horseradish peroxidase is about twice that for similarly sized dextran (∼40 kDa), suggesting that alveolar epithelium segregates the passage of macromolecules by shape (i.e., globular proteins vs. long-chained sugars of the same molecular weight). In contrast, P app of 3.7 × 10−10 cm/s for horseradish peroxidase was reported for filter-grown Madin-Darby canine kidney cell monolayers (with transepithelial resistance of ∼2,000 Ωcm2) (11). Horseradish peroxidase translocation across tight rat alveolar epithelial cell monolayers resulted in transepithelial pinocytotic rate of ∼0.5 nl/(cm2/min) in either apical-to-basolateral or opposite direction. This estimation of transalveolar epithelial pinocytic rate is insignificant compared with water absorption that is osmotically coupled to active Na+removal across the alveolar epithelial barrier of ∼15.6 nl/(cm2/min), suggesting that pinocytosis is not the major contributor for fluid resorption across the respiratory epithelial tract of the distal lung, although it may be important for protein transport. In line with this concept, recent evidence suggests that water channels (such as aquaporin-5 expressed in apical plasmalemma of type I, but not type II, cells) are likely to be important in osmotic water resorption across the alveolar epithelium (117), although regulation of pinocytosis across alveolar epithelium under pathophysiological conditions remains to be determined.
In an effort to delineate the routes and mechanisms for macromolecule transport across alveolar epithelium, P app for dextrans (of different molecular masses) was determined utilizing standard tracer techniques coupled with high-performance liquid chromatography for identification of intact and degraded dextrans.P app values of fluorescein isothiocyanate (FITC)-labeled dextrans estimated in the apical-to-basolateral and opposite directions were not significantly different from each other, regardless of molecular size (4, 10, 40, 70, and 150 kDa), across tight rat alveolar epithelial cell monolayers (74). Transport rates of dextrans appear to be dependent on molecular size, as larger dextran molecules exhibited much smaller P app. Dextran transport rates decreased gradually up to 40 kDa (withP app ranges from 1.53 × 10−8to 0.13 × 10−8 cm/s), whereas larger dextrans of 70 and 150 kDa showed about the same P app of 0.13 × 10−8 cm/s, suggesting that dextrans with molecular masses >40 kDa predominantly opted for nondiffusional pathways (e.g., pinocytosis). In support of this hypothesis, lowering experimental temperature from 35°C to 4°C led to ∼50% decrease in P app for dextrans up to 40 kDa, consistent with predominant diffusional movement of the smaller dextrans. In contrast, the temperature-dependent decrease inP app for 70- and 150-kDa dextrans was close to 90%, indicating that these larger dextrans preferentially traversed alveolar epithelium via nondiffusional transport pathways (e.g., via pinocytotic pathways). Dextrans did not show much degradation during transit across the alveolar epithelium (74). Equivalent pore analysis, based on P app of the smaller dextrans (i.e., 4–40 kDa that appear to be diffusion limited) and other hydrophilic solutes, yielded a pore radius of ∼6 nm. These data suggest that hydrophilic molecules with a radius >6 nm are excluded from passive diffusional pathways (i.e., passive leak via paracellular routes) of the alveolar epithelial barrier.
Absorption of Ovalbumin and Transferrin Across Rat Alveolar Epithelial Cell Monolayers
Unidirectional fluxes of several radiolabeled proteins (e.g., ovalbumin and transferrin) across rat alveolar epithelial cell monolayers exhibited asymmetry, resulting in net absorption (76). Moreover, P app of these proteins were about two to three orders of magnitude greater than those reported for 40- and 70-kDa dextrans (74, 75), suggesting the involvement of nondiffusional, transcytotic processes for absorption of these proteins across the monolayers. Detailed transport mechanisms and routes for alveolar epithelial transport of ovalbumin and transferrin remain unknown.
Characteristics of Albumin Transport Across Rat Alveolar Epithelial Cell Monolayers
Studies showed that unidirectional fluxes of [3H]- methylated BSA (∼67 kDa) were asymmetric and about two to three orders of magnitude greater than those of 70-kDa dextrans (74, 75). The radiolabel flux in the apical-to-basolateral direction across open-circuited rat alveolar epithelial cell monolayers was twice that in the opposite direction. P app for [3H] BSA was 2.06 × 10−7 cm/s and 1.13 × 10−7 cm/s (P < 0.05) in apical-to-basolateral and opposite directions, respectively. The apical and basolateral downstream fluids contained 50 and 85% intact albumin, respectively (76). These asymmetric and rapid fluxes of albumin are not likely secondary to methylation of lysine groups in the albumin surface, since labeling albumin with FITC led to similar flux rates and fractions of intact albumin (K.-J. Kim, unpublished data).
The saturable net absorption of albumin across the in vitro model of alveolar epithelium is mediated by specific albumin-binding protein(s) expressed in the pneumocytes. Endothelial gp60 (60-kDa albumin-binding glycoprotein) antibody (100, 114, 115, 118, 119) cross-reacts with membrane gp60 of rat alveolar epithelial cells (61, 66). With the use of albumin-tagged photoaffinity probe, an apical membrane protein of ∼60 kDa was found from cultured alveolar epithelial cells (Ref. 66 and S. M. Vogel and A. B. Malik, unpublished observation), whereas gp30 and gp18 (i.e., endothelial scavenger-type receptors) were not detected by the same albumin photoaffinity probe (S. M. Vogel and A. B. Malik, unpublished observation), suggesting that these scavenger-type receptors do not bind to the unmodified albumin used in these studies. Albumin absorption across the alveolar epithelial barrier may be mediated by gp60 albumin-binding protein in alveolar epithelial cells. Caveolae-like pits and numerous other vesicles are also present in type I cells of the distal air spaces of the lung (16, 30, 61, 64, 85,91), suggesting that caveolae-mediated protein internalization also plays a role in alveolar epithelial protein translocation, as has been reported in endothelium (82, 83). Thus the presence of specific receptors for albumin in alveolar epithelial cells as opposed to nonspecific adsorption is a prerequisite for net absorption of albumin across the epithelial barrier.
We recently demonstrated in cultured type II alveolar epithelial cells that albumin transcytosis is regulated by gp60 (61). Type II cells internalized fluorescently labeled albumin and anti-gp60 antibody, which colocalized in the same plasmalemma vesicles. In addition, antibody (100 μg/ml) cross-linking of gp60 for brief periods (15 min) markedly stimulated vesicular uptake of fluorescently tagged albumin. Cross-linking of gp60 with its antibody has also been used in cultured bovine pulmonary microvessel endothelial cells in which it enhanced albumin transcytosis via activation of Src protein tyrosine kinase (114, 118, 119). The caveolar disrupting agent, filipin (10 nM), abolished the stimulated internalization of albumin in alveolar epithelial cells. Prolonged stimulation of type II cells (>1 h) with cross-linking anti-gp60 antibody produced the loss of cell surface gp60 and abolished albumin uptake.125I-albumin was also instilled into distal air spaces of lungs, and resulting 125I-albumin efflux into the vascular perfusate was determined (61). Unlabeled albumin (studied up to 10 g/100 instilled ml) inhibited 40% of the transport of labeled albumin with an IC50 value of 0.34 g/100 ml. Filipin blocked this displacement-sensitive component of125I-albumin transport but had no effect on the transport of the paracellular tracer [3H]mannitol. Displacement-sensitive 125I-albumin transport had significantly greater Q10 than the nondisplaceable component. Moreover, cross-linking of gp60 by antibody instillation stimulated only the displacement-sensitive 125I-albumin transport across respiratory epithelial barrier in intact rat lungs. These results indicate the obligatory role of gp60 in the transcytosis of albumin across the distal air-blood (mostly alveolar epithelial) barrier.
Net saturable absorption of albumin across tight rat alveolar epithelial cell monolayers appears to occur predominantly via transcytosis, since albumin fluxes decreased by up to ∼95% in either apical-to-basolateral or opposite direction at 15°C (i.e., resulting in net absorption not significantly different from 0), and the fluxes observed at 15°C and 4°C were not significantly different from each other (66). It is highly unlikely that albumin traverses the normal alveolar epithelium predominantly via passive diffusion. Translocation of intact albumin across the alveolar epithelial barrier via nonspecific adsorptive endocytosis is also unlikely because the net negative charge on cell plasma membranes, as decorated by the cationic molecule ferritin (123), would be expected to hinder cell surface adsorption of the negatively charged albumin at physiological pH. Albumin internalized by fluid-phase endocytosis did not require adsorption of ligands and may only account for <1% of total intact albumin flux based on the rate of alveolar epithelial pinocytosis estimated with horseradish peroxidase as the fluid-phase marker (75). Moreover, the amount of pinocytosed albumin is expected to increase linearly as upstream albumin concentration is increased, but it is not likely to be transcytosed intact across the monolayer because pinocytosed cargo is largely degraded at lysosomes (11, 75).
IgG Transport Across Rat Alveolar Epithelial Cell Monolayers
IgG has been shown to be present in BAL fluid, although underlying transport mechanisms are unknown. By contrast, IgG transport across several epithelial barriers (mammary, neonatal intestine, placenta, and yolk sac) has been demonstrated (27, 37, 58, 68, 69, 80, 95, 103,104, 109), which is thought to take place via an IgG binding receptor, FcRn. Endothelial cells also express FcRn that plays an important role in degradation of excess IgG under pathological conditions (14). Although lung is known to express FcRn mRNA (102), the exact locale and its relative abundance in various cells in the lung are unknown.
We recently reported preliminary data on IgG transport across primary cultured rat alveolar epithelial cell monolayers (39) showing that unidirectional fluxes (J) of biotinylated rat IgG (biot-rIgG) saturated with increasing concentrations of rIgG. Lowering temperature from 35°C to 4°C resulted in major reductions of biot-rIgG J ab (apical-to-basolateral flux) and J ba (opposite flux), respectively. The biot-rIgG J ab decreased markedly in the presence of excess unlabeled rat Fc [but not Fab, F(ab′)2, or IgY] in upstream fluid (39). The expression of FcRn, an endothelial/epithelial type Fc receptor, in rat alveolar epithelial cell monolayers was confirmed by RT-PCR and Northern analysis (38). These data indicate that alveolar epithelial IgG transport most likely occurs via FcRn-mediated transcytosis.
Secretion of Polymeric Immunoglobulins, Such as IgA, Across Rat Alveolar Epithelial Cell Monolayers
The presence of dimeric IgA (dIgA) in the airways of the lung has been well documented (17, 81, 110). The levels of secretory IgA (sIgA) in BAL fluid and sputum were reported to be affected in a number of inflammatory diseases (81, 110). For example, the sIgA level in BAL fluid was elevated in asthmatic patients compared with healthy controls. A mean coefficient of excretion in human BAL fluid for dIgA (relative to albumin) of 20 was reported, which is comparable to the value of 22 found in human hepatic bile but substantially lower than that in jejunal fluids and saliva (218 and 354, respectively) (25, 26). sIgA is present in alveolar lining fluid and is especially prominent in surfactant (86). Lung cells expressing pIgR have been demonstrated using immunohistochemical techniques from the trachea through the terminal bronchioles by some investigators (49), whereas other investigators using similar approaches concluded that positive cells are absent in respiratory bronchioles, alveolar duct, and alveolar epithelium (93, 111). These conflicting reports may be due to relatively lower levels of pIgR expression in distal respiratory epithelial tract compared with airways, including bronchial trees, as pIgR expression has been reported for both nonciliated cells of bronchioles (including respiratory bronchioles) and type II pneumocytes, albeit at low levels (51). In contrast, airway goblet cells do not appear to express pIgR (51). These reports suggest that alveolar epithelium contributes to mucosal defense of the distal air spaces of the lung by secreting sIgA via pIgR-mediated transcytosis.
We reported preliminary data on pIgR expression in primary cultures of rat alveolar epithelial cell monolayers (36, 71). When cultured rat alveolar epithelial cells were pulse labeled with [35S]methionine/cysteine and then chased for various time periods, a glycosylated protein of ∼100 kDa was found in the cells, while a radiolabeled protein of ∼80 kDa was secreted into apical (but not basolateral) bathing media of alveolar epithelial cell monolayers (36). The appearance of the 80-kDa protein in the apical bathing fluid coincided with the loss of the 100-kDa protein from cells. In addition, we confirmed the expression of pIgR mRNA of ∼3.9 kb in primary cultured rat alveolar epithelial cells by RT-PCR and subsequent Northern analysis (126). These data indicate that alveolar epithelial cells synthesize pIgR and secrete secretory component (SC). Net secretion of SC (and pIgR-mediated secretion of pIgA/IgM) into alveolar lining fluid may play important roles in mucosal defense of the distal region of the lung, in part supporting the function of alveolar macrophages.
SUMMARY AND CONCLUSIONS
In summary, alveolar epithelial protein transport appears to be protein specific in that some proteins are translocated across the air-blood barrier via receptor-mediated transcytosis (e.g., albumin, IgG, and pIgA), whereas other proteins may opt for nonspecific fluid-phase endocytosis (Fig. 1). Pinocytosed proteins appear to be degraded, resulting in very low and symmetric transport of intact molecules across the alveolar epithelial barrier. Translocation of large serum proteins (e.g., albumin, IgG) via paracellular routes by restricted passive diffusion does not appear to be the primary route, although under pathological conditions such passive diffusion may become the main route of protein leak.
Some important questions left unanswered include: 1) specificity of protein transport processes in type I vs. type II cells,2) presence/absence of transport processes specific for serum proteins other than albumin, IgG, and pIgA, 3) molecular identity of albumin binding site(s) (e.g., gp60) expressed in alveolar epithelial cells, 4) cellular signaling mechanisms involved in alveolar epithelial protein transport, and 5) regulatory mechanisms underlying alveolar epithelial transport in health and disease.
Helpful discussions and careful reading of the manuscript by Drs. Edward D. Crandall at USC Keck School of Medicine and Stephen Vogel at University of Illinois are duly acknowledged.
This work was supported in part by the Hastings Foundation, American Heart Association Grant-in-Aid 9950442N (K.-J. Kim), and National Institutes of Health Research Grants HL-38658 (K.-J. Kim), HL-45638 (A. B. Malik), HL-64365 (K.-J. Kim), and HL-60678 (A. B. Malik).
Address for reprint requests and other correspondence: K.-J. Kim, Dept. of Medicine, Rm. HMR 914, Univ. of Southern California Keck School of Medicine, 2011 Zonal Ave., Los Angeles, CA 90033 (E-mail:).
- Copyright © 2003 the American Physiological Society