Am J Physiol Lung Cell Mol Physiol 284: L247–L259, 2003;
10.1152/ajplung.00235.2002.
invited review
Protein transport across the lung epithelial barrier
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KWANG-JIN KIM1 AND ASRAR B. MALIK2
1
Departments of Medicine, Physiology, and Biophysics, Molecular Pharmacology and
Toxicology, and Biomedical Engineering, Will Rogers Institute Pulmonary Research
Center, Keck School of Medicine and Schools of Pharmacy and Engineering, University
of Southern California, Los Angeles, California 90033; and 2Department of
Pharmacology, College of Medicine, University of Illinois, Chicago, Illinois 60612
Kim, Kwang-Jin, and Asrar B. Malik. Protein transport across the
lung epithelial barrier. Am J Physiol Lung Cell Mol Physiol 284:
L247–L259, 2003; 10.1152/ajplung.00235.2002.—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 clathrincoated 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; albumin; secretory component; immunoglobulin G
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
MACROMOLECULE TRANSPORT ACROSS
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:
[email protected]).
http://www.ajplung.org
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 re-
1040-0605/03 $5.00 Copyright © 2003 the American Physiological Society
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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
AJP-Lung Cell Mol Physiol • VOL
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 instilled 125I-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 airblood 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
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stored 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.
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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
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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 acidsoluble 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
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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.
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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
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Fig. 1. Schematic diagram illustrating protein transport processes in the alveolar epithelial barrier. Discussed are
protein transport properties of an in vitro model of primary cultured rat alveolar epithelial cells that exhibit
morphological and phenotypic traits of in vivo type I cells. Specific receptors for albumin, i.e., 60-kDa albuminbinding glycoprotein (gp60 also known as albondin), immunoglobulin G (FcRn), and polymeric immunoglobulin A
(pIgR) are shown. Other protein receptors not shown include those for insulin and transferrin, both of which are
also expressed in basolateral membranes of alveolar epithelial cells. The specific albumin-binding protein (or
receptor), gp60, was first identified in the endothelial cells and later found also to be expressed in the alveolar
epithelial cells. Albumin is transported across alveolar epithelium via gp60- and caveolae (shown as pink
circles)-mediated transcytosis (illustrated by the open, red-outlined arrow showing direction of net absorption). IgG
appears to be similarly absorbed via FcRn-mediated transcytosis across the alveolar epithelium, shown in blue
receptors and open, blue-outlined arrow for direction of absorption. Secretory component (SC; i.e., the extracellular
portion of pIgR) appears to be secreted into alveolar lining fluid (green receptors and direction of secretion).
Question marks next to the receptors or processes denote the insufficient proof of receptor expression or existence
of such processes in type II cells. Type I cells and endothelial cells are known to contain numerous vesicles and
membrane invaginations (including caveolae and clathrin-coated pits) that are expected to play important roles in
internalization of proteins and transcellular movement of cargo proteins. In endothelium, caveolae structures
outnumber clathrin-coated pit structures by 19 to 1, whereas the relative number of these structures in alveolar
epithelial cells is not clear. Relative contribution of transcytosis (or endocytosis) mediated by caveolae vs.
clathrin-coated pits to overall serum protein transport across alveolar epithelium also remains unknown. It is also
unclear whether type II cells have the ability to transport proteins via caveolae-mediated process (e.g., involving
gp60 and other albumin-binding proteins). It is important to note that restricted passive diffusion of large serum
proteins (e.g., albumin, IgG, and pIgA) via the paracellular route (i.e., between epithelial cells) plays an
insignificant role in the net absorption or secretion of these proteins across normal alveolar epithelium. Under
pathological conditions where paracellular routes are deranged due to inflammation and injury, the paracellular
leak of those large proteins may predominate. Defining the signaling pathways regulating transcytotic processes
under physiological and pathological conditions remains a challenge of future studies.
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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 colonystimulating 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
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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
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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
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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 receptormediated (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 receptormediated 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 caveolincoated 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
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and molecular [e.g., progressive expression of T1␣,
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 ⍀cm2, and shortcircuit current of ⬃5 ␮A/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 (Papp) 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 decreased Papp for horseradish peroxidase
by ⬃80%, suggesting that horseradish peroxidase traversed primarily via nondiffusional routes across the
alveolar epithelium. Interestingly, Papp 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, Papp 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 ⍀cm2)
(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
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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 cellspecific 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)
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INVITED REVIEW
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, Papp of these
proteins were about two to three orders of magnitude
greater than those reported for 40- and 70-kDa dexAJP-Lung Cell Mol Physiol • VOL
trans (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 opencircuited rat alveolar epithelial cell monolayers was
twice that in the opposite direction. Papp 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 ␮g/ml) cross-linking of gp60 for
brief periods (15 min) markedly stimulated vesicular
uptake of fluorescently tagged albumin. Cross-linking
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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, Papp 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. Papp
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 Papp. Dextran
transport rates decreased gradually up to 40 kDa (with
Papp ranges from 1.53 ⫻ 10⫺8 to 0.13 ⫻ 10⫺8 cm/s),
whereas larger dextrans of 70 and 150 kDa showed
about the same Papp 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 Papp for dextrans up to 40 kDa,
consistent with predominant diffusional movement of
the smaller dextrans. In contrast, the temperaturedependent decrease in Papp 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 Papp 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.
L255
INVITED REVIEW
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, plaAJP-Lung Cell Mol Physiol • VOL
centa, 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 biotrIgG Jab (apical-to-basolateral flux) and Jba (opposite
flux), respectively. The biot-rIgG Jab 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
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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 of 125I-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).
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INVITED REVIEW
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 receptormediated transcytosis (e.g., albumin, IgG, and pIgA),
whereas other proteins may opt for nonspecific fluidphase 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).
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