Am J Physiol Lung Cell Mol Physiol 286: L679–L689, 2004;
10.1152/ajplung.00205.2003.
Invited Review
Mechanisms of alveolar protein clearance in the intact lung
Randolph H. Hastings,1 Hans G. Folkesson,2 and Michael A. Matthay3
1
Research and Anesthesiology Services, Veterans Affairs San Diego Healthcare System, San Diego 92161;
Departments of Medicine and Anesthesiology, Cardiovascular Research Institute, University of California,
San Francisco, California 94143-0130; and 2Department of Physiology and Pharmacology,
Northeastern Ohio Universities College of Medicine, Rootstown, Ohio 44272-0095
3
acute respiratory distress syndrome; endocytosis; diffusion; protein transport pulmonary edema
from the alveolar space is an
important process in recovery from pulmonary edema. Albumin and immunoglobulin G (IgG) are present in pulmonary
edema fluid in concentrations that are 40–65% of plasma levels
in hydrostatic pulmonary edema and 75–95% in lung injury
pulmonary edema. Concentrations of albumin, for example,
may be 5 g/100 ml or more. Protein concentrations rise even
higher during the recovery phase from alveolar edema because
the salt and water fraction of edema fluid is cleared much faster
than albumin and IgG (6, 37, 58, 59, 72). High protein
concentrations are significant because increased protein osmotic pressures slow alveolar fluid clearance. For example,
soluble protein concentrations in alveolar liquid rose from
5.9 ⫾ 0.4 g/100 to 10.2 ⫾ 1.2 g/100 ml over 12 h after
instillation of autologous serum into the distal spaces of sheep
lungs, and calculated protein osmotic pressure in alveolar
liquid increased from 40 to 53 cmH2O (57). Alveolar liquid
clearance slowed from 8%/h in the first 4 h to 3%/h at the end
CLEARANCE OF SERUM PROTEINS
Address for reprint requests and other correspondence: R. H. Hastings, VA
Medical Center (125), 3350 La Jolla Village Dr., San Diego, CA 92161-5085
(E-mail: [email protected]).
http://www.ajplung.org
of this period. In addition to limiting liquid clearance, highly
concentrated alveolar protein may precipitate, necessitating
clearance of insoluble as well as soluble protein. Inability to
clear alveolar protein may play a role in poor outcomes after
pulmonary edema. Patients dying with acute lung injury and
the acute respiratory distress syndrome (ARDS) have large
quantities of insoluble protein in their air spaces, and nonsurvivors of lung injury after ARDS have three times as much
protein in their alveoli as survivors (1, 13). Accordingly, the
mechanism of alveolar protein clearance is a physiologically
and clinically significant problem.
Several mechanisms have been proposed for the removal of
protein from the alveoli, including clearance by the mucociliary escalator, phagocytosis by macrophages, intra-alveolar
catabolism, passive diffusion between cells in the epithelial
barrier, and endocytic transport across the epithelial cells in
vesicles, a process known as transcytosis. Wang et al. (83)
recently showed that liquid microinjected into small groups of
surface alveoli of gas-inflated lungs redistributed within seconds to adjacent alveoli. This movement appeared to be convective flow because it was unaffected by inhibitors of active
fluid transport. Convection from alveoli to distal airways could
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Hastings, Randolph H., Hans G. Folkesson, and Michael A. Matthay.
Mechanisms of alveolar protein clearance in the intact lung. Am J Physiol Lung Cell
Mol Physiol 286: L679–L689, 2004; 10.1152/ajplung.00205.2003.—Transport of
protein across the alveolar epithelial barrier is a critical process in recovery from
pulmonary edema and is also important in maintaining the alveolar milieu in the
normal healthy lung. Various mechanisms have been proposed for clearing alveolar
protein, including transport by the mucociliary escalator, intra-alveolar degradation,
or phagocytosis by macrophages. However, the most likely processes are endocytosis across the alveolar epithelium, known as transcytosis, or paracellular diffusion
through the epithelial barrier. This article focuses on protein transport studies that
evaluate these two potential mechanisms in whole lung or animal preparations.
When protein concentrations in the air spaces are low, e.g., albumin concentrations
⬍0.5 g/100 ml, protein transport demonstrates saturation kinetics, temperature
dependence indicating high energy requirements, and sensitivity to pharmacological agents that affect endocytosis. At higher concentrations, the protein clearance
rate is proportional to protein concentration without signs of saturation, inversely
related to protein size, and insensitive to endocytosis inhibition. Temperature
dependence suggests a passive process. Based on these findings, alveolar albumin
clearance occurs by receptor-mediated transcytosis at low protein concentrations
but proceeds by passive paracellular mechanisms at higher concentrations. Because
protein concentrations in pulmonary edema fluid are high, albumin concentrations
of 5 g/100 ml or more, clearance of alveolar protein occurs by paracellular
pathways in the setting of pulmonary edema. Transcytosis may be important in
regulating the alveolar milieu under nonpathological circumstances. Alveolar
degradation may become important in long-term protein clearance, clearance of
insoluble proteins, or under pathological conditions such as immune reactions or
acute lung injury.
Invited Review
L680
ALVEOLAR PROTEIN CLEARANCE
AJP-Lung Cell Mol Physiol • VOL
important and their relative quantitative contributions under
different conditions. The article will examine several characteristics that may differentiate the two mechanisms: size dependence, saturation kinetics, temperature dependence, and
sensitivity to agents that manipulate the rate of endocytosis.
The review will also briefly consider cell mechanisms of
clearance of insoluble protein from the distal air spaces of the
lung in vivo.
VESICLES VS. CHANNELS
Bignon and colleagues (7, 8) were the first of several
investigators (17, 46) to report that alveolar type I and type II
cells contained endogenous albumin and immunoglobulins in
intracellular vesicles. Micrographs of protein in vesicles demonstrate that the alveolar epithelium is capable of endocytosis
of serum protein but do not establish whether the protein is
transported across the epithelium, indicate the direction of
transport, nor quantify how much protein is transported. Vesicles do not necessarily transport their contents across a barrier.
Protein in vesicles could be returned to the alveolar space with
no net transport across the epithelium, akin to type II cell
recycling of surfactant phospholipid between intracellular and
extracellular compartments (34). Williams (87) found that type
II cells transported cationic ferritin from alveolar liquid to
organelles of the secretory pathway, supporting the operation
of recycling routes in the lung. Microscopy studies also provide no information about how the protein entered the alveolar
vesicles. More recent work suggests that albumin may undergo
receptor-mediated uptake through binding to gp60 on alveolar
epithelial cells. John and colleagues (46) have shown that
cultured pneumocytes internalize fluorescent albumin into
plasmalemmal vesicles together with gp60 and that albumin
and gp60 are located in caveolae, vesicles coated with the
protein caveolin. Furthermore, cross-linking gp60 with antigp60 antibodies and secondary antibodies stimulates endocytosis of gp60 and albumin, whereas prolonged stimulation with
the cross-linking antibody depletes the type II cell surface of
gp60 and abolishes endocytic albumin uptake. Albumin undergoes receptor-mediated transcytosis bound to gp60 in pulmonary capillary endothelium (71). Thus gp60 could play a role in
alveolar albumin clearance. The evidence concerning this hypothesis is discussed in the section PHARMACOLOGICAL MANIPULATION OF ENDOCYTOSIS to follow.
The alveolar epithelium is a tight epithelium and is the
primary barrier restricting passage of solutes and water into or
out of the alveolar space. Consequently, some authorities have
questioned whether molecules as large as serum proteins can
pass through the epithelium through paracellular channels (77).
Direct microscopic observation of proteins passing through
paracellular passages in the epithelium has not been reported,
but the slow rate of transalveolar protein flux connotes that
channels of sufficient size to transmit large macromolecules
would be rare and exceedingly unlikely to be encountered by
electron microscopy. We can demonstrate this point with a few
simple calculations, using results from a study that modeled
large pore frequency in the lung. Conhaim and coworkers (17)
made theoretical calculations of the number and size of pores
necessary to support the observed transport rates of varioussized hetastarch molecules across the alveolar epithelial barrier
in rat lungs (Table 1). On the basis of their modeling work, we
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occur during initial alveolar flooding but would not necessarily
contribute to clearance from the lung (65). It has yet to be
studied as a means for alveolar protein clearance to our
knowledge. In contrast, the other mechanisms have received
considerable attention. For example, the rate of alveolar protein
clearance is unchanged by the presence or absence of a cuffed
endotracheal tube, indicating that the mucociliary escalator is
an insignificant route for escape of protein from the lung (57,
59). Only small amounts of alveolar tracer protein are found in
macrophages, and most alveolar protein reaches the bloodstream intact in the first few days after instillation of solutions
of protein in the air spaces (5, 33, 57), suggesting minimal
impact for these processes. The importance of the first three
mechanisms has been discounted in acute alveolar protein
clearance, as discussed in detail in previous review articles (25,
38, 56, 67). Thus paracellular diffusion and transcytosis across
the distal lung epithelium remain the two most likely mechanisms for clearance of soluble protein from alveolar edema
fluid in normal lungs and will be the focus of this review. Net
diffusion of protein from the air spaces to the interstitium
would be supported by the positive protein concentration
gradient between alveolus and plasma, established by more
rapid clearance of alveolar liquid than alveolar protein during
the resolution of pulmonary edema. Macrophages and catabolism could play a role in long-term clearance of precipitated
protein, which will be discussed in a later section.
A recent article by Kim and Malik (48) provided an excellent review of how transcytosis might apply to alveolar protein
clearance. The article focused primarily on studies that have
utilized monolayers of alveolar epithelial cells grown on permeable supports. After primary culture, alveolar type II epithelial cells form confluent monolayers that are joined by tight
junctions and exhibit apical-basal polarity similar to the epithelium in vivo. The cells differentiate into type I-like cells
within days and form a high resistance (⬎2,000 ohm-cm2)
monolayer, providing a reasonable in vitro model for transport
across the tight alveolar epithelium. Drs. Kim and Malik
discuss evidence in favor of receptor-mediated transcytosis of
albumin and IgG by rat alveolar epithelial monolayers, including asymmetric apical-basolateral vs. basolateral-apical transport, saturation, competition between labeled and unlabeled
protein, and temperature dependence (49, 53, 54). Transport of
albumin appeared to be mediated by gp60, the albumin glycoprotein binding protein (46), whereas FcRn, the IgG binding
receptor, could carry IgG (22). Alveolar epithelial monolayers
are advantageous for studying transcellular transport because
of their high resistance. However, they comprised only one of
the 40 or more cell types present in the distal lung, and they
model only the type II cells of alveolar epithelial barrier with
no components representing the endothelium or the bronchial
epithelium. Consequently, data from monolayer studies may
not present a complete picture of transport mechanisms active
in the lung in vivo.
This review will focus on studies of alveolar protein clearance in whole animal or whole lung models under predominantly normal conditions. We are most interested in how
clearance applies to the setting of resolution of alveolar edema.
The main question is: are plasma proteins cleared in vivo
predominantly by transcytosis or by paracellular pathways?
Evidence will be presented to demonstrate that both processes
operate. The problem is to determine which pathway is more
Invited Review
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ALVEOLAR PROTEIN CLEARANCE
Table 1. Theoretical volume occupied by one large pore in the alveolar epithelium vs. the volume examined
by electron microscopy
Theoretical Number of Large Alveolar Epithelial Pores
Frequency of pores/alveoli*
Alveoli/lung†
No. of pores/lung*
Alveolar Volume Containing One Pore
Alveolar epithelial volume (per one lung)‡
Volume/pore
Tissue Volume Sampled by Electron Microscopy
Section thickness*
Magnification (1 cm ⫽ 0.1 ␮m)*
Micrograph area
Tissue Area*
f ⫽ 1 pore/7 alveoli
A ⫽ 3.0 ⫻ 107
np ⫽ A ⫻ f ⫽ 4.3 ⫻ 106
Va ⫽ 0.161 ml
Va per pore ⫽ Va/np ⫽ 3.7 ⫻ 10⫺8 ml
Tissue volume examined
Large Pore Volume
Pore radius*
Pore length*
Pore volume
Epithelial Volume Containing One Pore versus Volume Examined by Microscopy
Va per pore vs. Vepith
Ratio
r ⫽ 17 nm ⫽ 1.7 ⫻ 10⫺6 cm
L ⫽ 0.7 nm ⫽ 7 ⫻ 10⫺8 cm
Vp ⫽ ␲ ⫻ r2 ⫻ L ⫽ 6.6 ⫻ 10⫺19 ml
3.7 ⫻ 10⫺8 ml vs. 4.3 ⫻ 10⫺13 ml
82,000:1
Va, alveolar volume; Vtiss, tissue volume; Atiss, tissue area; Vp, pore volume; Vepith, epithelial volume. *From Conhaim et al. (17); †From Tenney and Remmers
(78); ‡From Harris et al. (35).
estimate that one of these large pores would be found in the
alveolar epithelium on average in a volume of 3.5 ⫻ 10⫺10 ml
(per pore). For comparison, we next calculate the volume of
tissue that can be examined by electron microscopy. We use
the micrographs by Conhaim’s team to examine transport of
colloidal gold-labeled molecules in the lung epithelium as our
example. Based on section thickness and micrograph crosssectional area, each micrograph samples a tissue volume of
4.5 ⫻ 10⫺13 ml. The volume corresponding to each micrograph is nearly a million times greater than the volume of one
pore, so pore volume has little or no impact on how well the
pores are assessed by electron microscopy. To conclude the
problem, we note that the volume of alveolar epithelium
containing one pore is nearly 100,000 times greater than the
volume examined in one micrograph, 3.7 ⫻ 10⫺8 vs. 4.5 ⫻
10⫺13 ml. Thus surveying a region of the epithelium that
contained one of these pores might require taking tens of
thousands of micrographs. Even then, the pore might not be
readily apparent in the micrograph or might not be observed
passing a labeled protein molecule due to the orientation of the
pore. We conclude that electron microscopy has insufficient
sensitivity to discover or rule out paracellular transport of
macromolecules in the alveolar epithelium.
Most of the evidence in favor of paracellular alveolar protein
clearance comes from physiological studies on factors related
to diffusive or endocytic transport that will be described in later
sections of this review. However, a few tracer studies also
provide evidence in favor of passive diffusive transport. Conhaim and coworkers (15) filled isolated dog lobes with solutions containing a variety of tracers ranging in size from 1.2 to
405 nm in radius and measured concentrations in interstitial
liquid. The 405-nm fluorescent polystyrene particles were not
found in the interstitium, but 85 nm-radius latex beads and 95
nm-radius India ink particles crossed the epithelium. This
model could be subject to the criticism that the stress imposed
on lungs inflated to near total capacity with liquid could make
AJP-Lung Cell Mol Physiol • VOL
the epithelial barrier leaky. However, the investigators had
previously shown no difference in alveolar epithelial albumin
permeability in lungs inflated with low volumes compared with
lungs inflated to total lung capacity (16). Transcytosis of
particles 85–95 nm in radius is unlikely because they are so
large compared with the dimensions of endocytic vesicles.
Vesicles typically have radii on the order of 25–75 nm (75).
Williams (87) found some vesicles in the alveolar epithelium
with radii as large as 130 nm but reported that the radius of the
large majority of vesicles was ⬍90 nm. Hastings and coworkers (39) reported that alveolar epithelial vesicles averaged 35 ⫾
2 nm in radii. Thus Conhaim argued that large pores must be
present to provide a passage for their large tracers. These pores
were localized to the distal airway epithelium in the region of
the alveolar ducts, respiratory bronchioles, or their associated
alveoli (14). The presence of pores of adequate size shows that
paracellular passage of alveolar protein is possible but says
nothing about the contribution of this mechanism to alveolar
clearance of serum proteins. Physiological studies described
below help to delineate the relative importance of diffusion
versus transcytosis.
SIZE DEPENDENCE
The rate of absorption from the alveoli in vivo is size
dependent for most proteins. Effros and Mason (21) assembled
measurements of alveolar epithelial protein permeability from
many sources. The combined data demonstrated a consistent
inverse relationship between permeabilities and the corresponding molecular weights (MW) (Fig. 1). Theodore and
colleagues (79) reported that the alveolar epithelial permeabilities of sucrose, inulin, and dextran (60–90 kDa) in saline-filled
dog lungs varied inversely with molecular size and directly
with the free diffusion coefficient in water. Wangensteen and
Yankovich (84) found a larger permeability-surface area (PS)
product for sucrose than for albumin in rabbit lungs. Folkesson
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T ⫽ 100 nm ⫽ 1 ⫻ 10⫺5 cm
M ⫽ ⫻100,000
Areaphoto ⫽ 23.5 cm ⫻ 19 cm
Areatiss ⫽ (23.5 cm/M) ⫻ (19 cm/M)
⫽ 2.35 ⫻ 10⫺4 cm ⫻ 1.9 ⫻ 10⫺4 cm
⫽ 4.5 ⫻ 10⫺8 cm2
Vtiss ⫽ T ⫻ Areatiss ⫽ 4.5 ⫻ 10⫺13 ml
Invited Review
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ALVEOLAR PROTEIN CLEARANCE
and colleagues (27, 28, 30) found that the absorption of
intratracheally instilled proteins and peptides in unanesthetized
rats and pigs varied inversely with MW. In a study of pulmonary absorption of different-sized fluorescein isothiocyanate
(FITC)-labeled dextrans, Ohtani and colleagues (66) found that
rate of absorption decreased with increasing molecular size.
Conhaim and colleagues (17) found that transport of differentsized hetastarch molecules out of the alveoli could be modeled
by a system of epithelial pores of at least two sizes, 5 and 17
nm in radius, allowing size-dependent variation in the rate of
passage. Hastings and colleagues (37) reported an inverse
relationship between the Stokes radius and the amount of
radiolabeled protein tracer that remained in the lungs of anesthetized rabbits 8 h after instillation. These investigators also
examined size selectivity of protein clearance humans during
the resolution phase of pulmonary edema. The fraction of total
protein concentration made up of albumin was greater in
edema fluid than in plasma initially, consistent with sizedependent sieving of protein during edema formation (37). In
patients who showed resolution of the edema in the ensuing
hours, the albumin fraction decreased with time. This observation meant that albumin was being cleared more rapidly than
the other protein in edema fluid. These other proteins for the
most part would be proteins such as immunoglobulins that are
larger than albumin. Thus rat, rabbit, dog, pig, and human
studies provide evidence that protein clearance from the air
spaces of the lung is size dependent (17, 27, 28, 30, 37, 84).
Not all proteins, however, display the inverse size-clearance
relationship demonstrated by the forgoing work. Some proteins
are cleared from the lung more rapidly than expected for their
size. For example, the plasma concentration-time curves obtained for human growth hormone (hGH) (MW ⫽ 22,000)
after intratracheal instillation in rats demonstrated a more rapid
clearance from the respiratory tract than expected compared
with clearance of albumin (24). Surprisingly rapid clearance
also held true if hGH was aerosolized into the lung (68, 69).
These observations suggest that hGH may utilize a different
transport mechanism than other proteins. Because the rate is
faster than expected and is subject to saturation kinetics (discussed in the next section), the mechanism is probably receptor-mediated endocytosis.
AJP-Lung Cell Mol Physiol • VOL
SATURATION KINETICS
When receptors become fully occupied by ligand, increases
in concentration do not increase the rate at which ligand is
taken up by endocytosis. Thus lung protein clearance by
receptor-mediated endocytosis or transcytosis should exhibit
saturation kinetics. hGH clearance demonstrates this relationship (24). The appearance of hGH in the blood circulation after
lung instillation increases nonlinearly with instilled dose (Fig.
2A) (24), consistent with clearance, at least in part, by a
receptor-mediated mechanism. Albumin transport across the
upper airway epithelium follows saturation kinetics also and
may therefore depend on an endocytic transport process (47).
The half-maximal flux across bronchial epithelium occurs at
⬃1 g/100 ml albumin. However, unlike the situation for
passage across upper airway epithelium, saturation kinetics do
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Fig. 1. Alveolar protein clearance is size dependent for a wide range of
proteins over a wide range of molecular weights. Plot of data collected by
Effros and Mason (21) from many sources showing inverse relation between
rate of lung clearance and molecular weight over a wide range. [From Hastings
et al. (37).]
What does size-dependent alveolar protein clearance mean
relative to mechanism? Simply put, diffusive paracellular
transport, fluid phase and receptor-mediated transcytosis may
differ in the influence of molecular size. Variation relative to
size is a hallmark of diffusive transport. Diffusion is related to
size because diffusivity varies inversely with molecular mass.
Diffusion through water-filled channels shows additional size
effects for particles whose diameter approaches the diameter of
the channel, a phenomenon referred to as restricted diffusion.
The rate of transport by endocytosis, on the other hand, should
not necessarily be size dependent. Fluid-phase endocytosis
begins with vesicles formed from caveolae, clathrin-coated pits
that close around fluid adjacent to the cell. The protein concentration in the vesicle would be expected to match the
concentration of the outside fluid regardless of size, unless
other factors such as unstirred layer effects or steric hindrance
to caveolar access imparted sieving properties. Experimental
studies support independence of size for fluid phase endocytosis in the distal lung epithelium. Matsukawa and colleagues
(52) reported that 70-kDa dextran and 150-kDa dextran particles undergo pinocytosis across alveolar epithelial monolayers
at the same rate, whereas Williams (87, 88) observed that
vesicles in alveolar epithelial cells in vivo took up similar
amounts of 70-kDa dextran and two lectins with limited epithelial binding, conconavalin A (108 kDa) and Wisteria floribunda agglutinin (58 kDa).
There are reasons to expect that the rate of receptor-mediated endocytosis should not depend on size either. Receptor
binding can concentrate proteins in the vesicle above concentrations in the extracellular fluid, so the transport rate may be
greater than expected for ligand size, as is the case for hGH
(24, 68, 69) and other proteins. For example, vasoactive intestinal peptide (VIP, MW 3,450), which undergoes receptormediated endocytosis, appears to escape the lungs with a
half-time of 19 min, nearly as fast as pertechnetate (MW 163)
at 10 min and almost 10 times faster than diethylene triamine
pentaacetate (MW 492), a much smaller molecule (3). The
calculated kinetic constant for VIP is 3.7 ⫻ 10⫺2 min⫺1, nearly
an order of magnitude greater than would be predicted for its
size (see Fig. 1). Thus the observation that albumin and IgG
permeate the alveolar epithelial barrier at a rate expected for
their size is consistent with a paracellular mechanism but
would not necessarily be anticipated for clearance by transcytosis.
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ALVEOLAR PROTEIN CLEARANCE
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not hold for clearance of serum proteins out of the alveoli over
comparable protein concentrations. For example, clearance of
instilled albumin is proportional to concentration between 3
and 25 g/100 ml (Fig. 2B) (28), and clearance of 125I-albumin
is unaffected by unlabeled albumin between 5 and 10 g/100 ml
(46). The failure to observe saturation kinetics argues against a
receptor-mediated mechanism for absorption of large quantities of serum proteins out of alveolar edema fluid.
Receptor-mediated absorption of albumin may be important
in the distal air spaces at lower albumin concentrations. John
and coworkers (46) compared the PS product for transport of
125
I-albumin out of the alveolar space of isolated perfused rat
lungs with no unlabeled albumin and with total unlabeled
albumin concentrations from ⬃0.001 to 10 g/100 ml. The PS
product fell when unlabeled albumin was increased from 0 to
5 g/100 ml. At higher concentrations, the product was constant,
indicating that albumin clearance increased in proportion to
concentration. These results show that albumin transport occurred by two processes depending on concentration. A saturable component predominated at low albumin concentrations,
consistent with absorption mediated by cell surface albumin
binding sites. Unlabeled albumin competed for these binding
sites with a half-maximal inhibitor concentration of 0.3 g/100
ml. Nonsaturable albumin clearance at higher concentrations
was consistent with paracellular transport.
TEMPERATURE DEPENDENCE
The magnitude and pattern of temperature dependence distinguish between endocytic and diffusive transport. Diffusion
through aqueous channels slows as temperature decreases due
to increases in the viscosity of water and a decrease in the
diffusion coefficient. The rate of diffusion would decrease by
⬃40% for a fall in temperature from 37 to 17°C, assuming that
the dimensions of the channel and the molecule remained
constant. Temperature dependence is described conveniently
by the Q10, the ratio of the rates of a biological process with a
10°C change in temperature. The Q10 for diffusion would be
⬃1.6. On the other hand, endocytic transport requires energy
and should demonstrate a much greater dependence on temperature, generally a Q10 ⬎2. Furthermore, endocytic uptake is
AJP-Lung Cell Mol Physiol • VOL
abolished at a temperature between 10 and 20°C (63). Thus a
plot of the rate of transcytosis as a function of temperature
should show an inflection point somewhere in that range. In
contrast, diffusive transport should demonstrate a monotonic
relationship with temperature.
Hostetter and colleagues (41) aerosolized 125I-albumin, total
albumin concentration 0.02 g/100 ml, into isolated rabbit lungs
and compared clearance at two temperatures, 37 and 12°C.
Clearance decreased 89% between the two temperatures
(Q10 ⫽ 2.4), much more than the 40–50% decrease expected
for diffusion above. Thus these data suggested that transport of
low concentrations of albumin out of the alveolar space occurred by transcytosis.
John et al. (46) examined the effects of concentration and
temperature on tracer albumin clearance. The investigators
studied the effects of temperature on clearance of 0.05 g/100
ml albumin, representing saturable transport, and 5 g/100 ml
albumin, the nonsaturable component. At 0.05 g/100 ml albumin, the PS product for 125I-albumin decreased slightly over
50% when temperature was reduced from 37 to 27°C, a Q10 of
2.1. At 5 g/100 ml, the PS product fell by ⬃40%, a Q10 of 1.6.
Thus temperature sensitivity was greater at the lower albumin
concentration, corresponding to saturable transport (see previous section SATURATION TRANSPORT), than at the higher concentration corresponding to nonsaturable albumin clearance. In the
opinion of the authors, the temperature dependence at higher
concentrations and the absence of saturation were both consistent with albumin transport by a paracellular route. In summary, the Hostetter study and the John study both found
temperature sensitivity consistent with energy-dependent
transport, such as transcytosis, at low albumin concentrations,
0.02–0.05 g/100 ml. Temperature dependence at higher concentrations was consistent with a passive process, such as
paracellular diffusion.
PHARMACOLOGICAL MANIPULATION OF ENDOCYTOSIS
Effects of monensin and nocodazole on albumin transport.
Several investigators have evaluated the role of transcytosis in
alveolar protein clearance by attempting to increase or decrease
the rate of endocytosis in vivo with pharmacological agents.
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Fig. 2. Kinetics of lung to blood passage of human growth hormone (hGH) and bovine serum albumin (BSA). A: plasma levels
of hGH 24 h after instillation were nonlinearly related to intratracheal dose over a range of 36–720 ␮g/kg body wt in adult rats.
[Reproduced by permission of the Society of the European Journal of Endocrinology from Folkesson et al. (24).] B: plasma albumin
levels were nonlinearly related to dose at low concentrations of instilled albumin between 0 and 37 mg/kg. In contrast to low-dose
albumin and to hGH, plasma appearance was directly related to instilled load at higher doses, between 37 and 200 mg/kg. These
doses correspond to albumin concentrations in the instilled liquid of 3.8–20 g/100 ml. [From Folkesson et al. (28).]
Invited Review
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ALVEOLAR PROTEIN CLEARANCE
Hastings and colleagues (39) studied the effects of monensin
and nocodazole on alveolar clearance of albumin and IgG in
anesthetized rabbits. The rationale for this study was that
inhibiting endocytosis should block alveolar protein clearance
if endocytosis is the major pathway. Monensin is an ionophore
that interferes with vesicular Na⫹/H⫹ exchange. It blocks
acidification of endocytic compartments and disrupts recycling
of membrane components to the plasma membrane, thus inhibiting endocytosis (61, 76). Nocodazole reversibly disrupts
microtubules, which are necessary for vesicle translocation
across the cell. It inhibits transcytosis in Madin-Darby canine
kidney epithelial cells, Caco-2 intestinal epithelial cells, and
tracheal epithelial cells (12, 19, 55).
The investigators performed four control experiments to
demonstrate that monensin and nocodazole were active in
whole lung and that they effectively reduced endocytosis: 1)
electron microscopy with morphometric techniques demonstrated that the inhibitors increased the numerical density of
vesicles in the alveolar epithelium and capillary endothelium.
Thus the two drugs increased the size of internal membrane
compartments as expected if membrane traffic were disrupted.
2) The inhibitors were studied for effects on clearance of
surfactant apoprotein A (SP-A) from the alveolar space. SP-A
Fig. 4. Albumin uptake from the alveolar space across airway epithelium. Shown are lung sections immunohistochemically stained
for human albumin after instillation of albumin plus DMSO vehicle (A) or albumin plus nocodazole (B). Labels mark the alveolar
region (a) and a bronchus (b) and indicate the presence or absence of immunoreactive human albumin (⫹ or ⫺). Nocodazole
reduced uptake of immunoreactive human albumin by the alveolar parenchyma (B) compared with uptake in untreated control lung
(A), suggesting inhibition of endocytic uptake by the alveolar epithelium. However, albumin immunoreactivity was present in larger
airways regardless of treatment with DMSO or nocodazole (B). Because endocytosis was inhibited, the bronchial uptake may
represent paracellular transport of albumin into the interstitium. The upper airway epithelium is thought to contain large pores in
the region of the alveolar ducts or respiratory bronchioles (15). This micrograph suggests that these pores could support diffusive
clearance of serum proteins from the alveolar space. [Modified from Hastings et al. (39).]
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Fig. 3. Monensin interferes with surfactant apoprotein A (SP-A) endocytosis.
The chart shows recoveries of 125I-SP-A in rabbit lung and lavage liquid 2 h
after instillation. Lavage recoveries represent tracer still present in the air
spaces, while lung recoveries are the amount of tracer taken up by alveolar
epithelial cells or transported into the interstitium. Monensin increased the
amount of SP-A in the lung, indicating that it interfered with dissociation of
SP-A from its receptor, interrupted recycling of endosomal or vesicular
membrane to the cell surface, or both. In either case, the data indicate
inhibition of endocytosis. *P ⬍ 0.05 vs. control. [From Hastings et al. (39).]
is a positive control for endocytosis inhibition because type II
cells remove SP-A from the air spaces by receptor-mediated
endocytosis. SP-A binds selectively to the apical surface of
type II cells in a dose-dependent and saturable fashion (70, 89).
After internalization, the protein dissociates from its receptor
and progresses through vesicles, endosomes, multivesicular
bodies, and lamellar bodies (70, 90). Eventually, SP-A is
recycled back to the alveolar space (73, 90). Monensin increased the quantity of SP-A associated with the lung tissue
after 2 h (Fig. 3). Thus monensin inhibited recycling of SP-A
from the epithelial cells to the air spaces by either blocking
dissociation from the receptor or interfering with intracellular
sorting. In either case, the action was consistent with the
pattern expected for a compound that inhibited endocytosis (3).
Endocytosis of FITC-labeled albumin uptake was studied in
cultured alveolar epithelial cell monolayers. Monensin and
nocodazole reduced uptake by 20–50% over 4- and 8-h experiments, verifying that they were active against alveolar epithelial cells (4). Immunohistochemical techniques were used to
evaluate the effect of inhibitors on parenchymal uptake of 5
g/100 ml human albumin from the air space. Albumin immunoreactivity appeared in the epithelium in the absence of
inhibitors but was not found in alveolar epithelium treated with
monensin or nocodazole (Fig. 4). Because the inhibitors would
not block paracellular transport, monensin and nocodazole
must have blocked endocytic uptake of albumin. In summary,
the control experiments demonstrate that the two drugs blocked
transport by cultured alveolar epithelial cells and disrupted
alveolar membrane traffic in whole lung. Furthermore, monensin and nocodazole reduced transport of SP-A and albumin,
indicating that they inhibited endocytosis of protein by the
alveolar epithelium in vivo.
The studies were completed by investigating the effects of
the endocytosis inhibition on alveolar clearance of 125I-IgG
and 131I-albumin. The tracers were instilled into the lungs in
autologous plasma. Thus concentrations of unlabeled protein
were high, approximately equivalent to concentrations in lung
injury pulmonary edema fluid. There were no significant differences in the quantities of the two tracer proteins recovered
in lung or appearing in the plasma among control animals and
animals receiving monensin or nocodazole (Fig. 5). In other
words, alveolar clearance of albumin and IgG occurred at the
Invited Review
ALVEOLAR PROTEIN CLEARANCE
L685
same rate whether or not alveolar endocytosis was inhibited.
Therefore, a pathway other than transcytosis, possibly paracellular transport, must be important for clearing of serum protein
in high concentrations from the alveolar space in normal lungs.
A possible location for this pathway was suggested by the
micrograph from a lung treated with nocodazole in Fig. 4B.
The nocodazole had inhibited albumin uptake by the parenchyma, but albumin immunoreactivity was still present in the
interstitium of a small airway. Nocodazole is known to inhibit
albumin endocytosis by airway epithelium (19). Thus the
albumin must have reached the airway interstitium by crossing
a paracellular channel in the airway epithelium proximal to the
alveolar barrier. Furthermore, no such channel was apparent at
the alveolar level. This observation is consistent with the report
of Conhaim et al. (15) that proximal airway epithelium contains pores large enough for passage of particles up to 94 nm
in radius.
Effects on transport of riboflavin-albumin vs. albumin. Wangensteen and colleagues (85) examined the effect of monensin
and nocodazole on alveolar transport of bovine serum albumin
(BSA), riboflavin-conjugated albumin (riboflavin-albumin),
and sucrose. Riboflavin-albumin was tested because many cells
possess riboflavin receptors (40), and the authors believed that
the conjugate might undergo receptor-mediated transcytosis
utilizing this receptor. In fact, tracer [3H]riboflavin-albumin
was cleared twice as fast as [3H]albumin when instilled into the
lungs of anesthetized rats in 0.5% unlabeled protein, indicating
that it was transported differently from unconjugated albumin.
In isolated perfused rabbit lungs, nocodazole and monensin
decreased the PS product of riboflavin-albumin by ⬎50%,
consistent with absorption by transcytosis mediated by riboflavin receptors. The PS product of sucrose, a marker for
paracellular transport, and the PS product for albumin were
unaffected by the endocytosis inhibitors. Thus transcytosis was
quantitatively important for riboflavin-albumin but did not play
an important role in alveolar clearance of 0.5% albumin. The
conclusion of this study was similar to the one reached by
Hastings et al. (39).
Stimulation and inhibition of gp-60-mediated albumin uptake. John et al. (46) used pharmacological interventions to
investigate the role of gp60 in alveolar clearance of albumin in
isolated perfused rat lungs. They incorporated the effects of
concentration in their experiments because they had demonAJP-Lung Cell Mol Physiol • VOL
strated the presence of saturable and nonsaturable albumin
transport pathways as discussed earlier. The experiments employed interventions both to stimulate and to inhibit gp60mediated endocytosis.
The intervention to stimulate endocytosis was to instill gp60
primary antibodies and secondary antibodies into the lung with
tracer 125I-albumin plus low and high concentrations of unlabeled albumin. This treatment crosslinks gp-60 and stimulates
endocytosis of albumin in cultured endothelial cells (60) and
cultured type II cells (46). At low unlabeled albumin concentrations, 0.05 g/100 ml, gp60 cross-linking stimulated 125Ialbumin PS product by 58%. At high concentrations, 5 g/100
ml, cross-linking had no effect. The intervention to inhibit
endocytosis was to instill intratracheal filipin, a lipophilic
polyene antibiotic that binds to cholesterol. Filipin prevents
gp60-activated vesicle formation in endothelial cells (60). In
cultured type II cells, filipin depletes caveolae, the vesicles that
carry gp60 and albumin, within minutes of treatment (46). In
the isolated lung experiments, filipin reduced the PS product
for 125I-albumin by 40% when the air space levels of unlabeled
albumin were low. The antibiotic did not affect clearance of
tracer albumin at high levels of unlabeled carrier, nor did it
alter the PS product for sucrose, a marker for paracellular
transport. Thus stimulation of endocytosis augmented alveolar
albumin clearance, and endocytosis inhibition reduced clearance at low albumin concentrations, conditions in which saturable transport predominates. Transport at high concentrations
was unaffected by manipulating the rate of endocytosis. On the
basis of the saturation kinetics, the temperature dependence
described earlier, and the effects of pharmacologically manipulating endocytosis, the authors proposed that alveolar albumin
permeation occurred by gp60-mediated transcytosis of albumin
at nonsaturating albumin concentrations. At higher concentrations, they suggested that the transport mechanism was a
paracellular pathway. The three studies, by John et al. (46),
Hastings and coworkers (37), and Wangensteen and colleagues
(85), are consistent with each other. All three found no effect
of endocytosis inhibitors on alveolar albumin clearance with
high concentrations of alveolar albumin.
John and coworkers provided data that allow calculation of
the approximate capacity of the transcytosis mechanism relative to the paracellular route. They measured clearance of
125
I-albumin at low and high carrier albumin concentrations.
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Fig. 5. Lung (A) and plasma (B) recoveries of 125I-IgG and 131I albumin in control (E), monensin (F), and nocodazole groups (‚)
8 h after instillation. Tracer proteins were instilled into the lung in autologous plasma, a carrier with high concentrations of
unlabeled albumin and IgG similar to those present in lung injury pulmonary edema fluid. Endocytosis inhibitors did not
significantly change lung clearance or plasma appearance of albumin and IgG over 8 h. Control studies, such as the experiment
shown in Fig. 3 and those described in the text, demonstrated that monensin and nocodazole inhibited endocytosis of protein by
the alveolar epithelium. The results indicate that protein clearance continues unabated when endocytosis is reduced and suggest that
a mechanism other than endocytosis is important for alveolar protein clearance. [From Hastings et al. (39).]
Invited Review
L686
ALVEOLAR PROTEIN CLEARANCE
Approximately 35% of the tracer albumin was cleared from the
lung in 1 h when the carrier albumin concentration was 0.05
g/100 ml, but the investigators estimated that only 25% was
cleared by the saturable route. Thus the capacity of the transcytosis pathway to clear albumin in 1 h was ⬃0.5 mg ⫻ 0.25 ⫽
0.125 mg. At 5 g/100 ml albumin, conditions saturating the
receptor-mediated process, 90% remained at the end of the
hour. In this case 50 mg ⫻ 0.1 ⫽ 5 mg was cleared from the
air spaces. Thus when high concentrations of albumin are
present in the alveolar space, as with pulmonary edema, transcytosis accounts for 2.5% of the total albumin clearance and
paracellular pathways for the other 97.5%.
ALVEOLAR DEGRADATION
proteins. The vast majority of studies indicate that ⬎95% of
the protein instilled into the distal air spaces of the normal lung
reaches the blood circulation intact (4, 5, 27, 28, 31, 33, 57).
However, peptidases and lysosomes could participate in metabolism of specific peptides or larger proteins, such as VIP,
gastrin, or insulin. VIP is cleared from the air spaces more
rapidly than expected for its size and is completely degraded
during transit across the pulmonary epithelium into the vascular spaces (3). Gastrin is also degraded in the passage from the
air spaces into blood (R. H. Hastings, unpublished data).
Although cultured pneumocytes degrade substantial quantities
of insulin (50), insulin is cleared intact at a rate consistent with
its size relative to larger proteins in rabbit lungs (37).
AJP-Lung Cell Mol Physiol • VOL
If proteins are not handled appropriately at the membrane,
they may become antigenic and trigger local and systemic
immune and inflammatory responses. Folkesson and colleagues (29) investigated whether a prior immunization with
BSA altered the alveolar epithelial permeability of a closely
related protein, human serum albumin (HSA). The investigators picked HSA as the bystander protein for estimating permeability because it was similar in most characteristics to
BSA, the antigen. Alveolar epithelial HSA permeability increased when animals with anti-BSA IgG in their air spaces
were challenged with BSA. Thus high levels of circulating
specific antibodies and antigen may influence alveolar epithelial permeability to protein in general. Other investigators have
suggested that a preceding immunization may reduce or even
totally inhibit the passage of the specific antigen from the
alveolar space to the systemic circulation (9–11, 32, 42, 80).
These effects may be attributed to increased degradation of the
antigen and/or reduced penetration across the epithelial barrier
due to the increased size of immune complexes compared with
antigen alone. Significant levels of protein are present in
bronchoalveolar lavage fluid after sensitive asthmatics are
exposed to allergen (23). One implication of increased nonspecific protein permeability after allergen challenge is that it may
expose sensitive asthmatics to other potential antigens, resulting in development of additional allergies unrelated to the
original disease.
INSOLUBLE PROTEIN CLEARANCE
This review has focused on the mechanisms for removal of
soluble protein from the distal air spaces of the lung. From a
clinical perspective, hydrostatic pulmonary edema and increased permeability pulmonary edema (acute lung injury)
represent the most important clinical settings in which large
quantities of soluble protein collect in the distal air spaces of
the lung. In the presence of severe acute lung injury, some of
the protein in the air spaces of the lung precipitates out of
solution and forms an insoluble matrix in conjunction with
extracellular matrix proteins and fibrin. Microscopically, the
insoluble protein is identified pathologically as hyaline membranes, a classic hallmark of diffuse alveolar damage in patients with acute lung injury. Recent work indicates that the
mechanism for accumulation of the insoluble protein components in the distal air spaces of the lung is probably a product
of both procoagulant and proinflammatory mechanisms that are
activated in the presence of acute lung injury (43, 86).
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IMMUNITY
Alveolar macrophages. Alveolar macrophages are well
known for their capacity to endocytose and degrade exogenous
protein and peptides (74). Their endocytic capacity is greater
than that of type II alveolar epithelial cells (36). However,
alveolar macrophages do not appear to play a major role in
clearing protein over 24–48 h from the alveoli of the normal
lung. For example, the number of alveolar macrophages increased by 10-fold over 24 h after autologous serum was
instilled into sheep lungs, but the rate of protein clearance
remained constant during this period (5, 57). Less than 10% of
the instilled 125I-albumin was associated with alveolar macrophages 24 and 48 h after the instillation (5). Alveolar macrophages may become important in alveolar protein clearance
after 48 h. Berthiaume and colleagues (5) followed alveolar
protein clearance in awake sheep for 6 days after instilling
125
I-albumin into the lungs. The influx of macrophages into the
air spaces increased substantially between the second and sixth
days. Increased quantities of protein tracer were associated
with macrophages by the fourth day, and phagolysosomes with
staining characteristics similar to that of the instilled serum
were present in the macrophage cytoplasm. On the sixth day,
the quantities of protein-free 125I in lung lavage, urine, and
feces increased four- to fivefold compared with the quantity at
48 h. Thus as time progressed, resident cells in the air spaces
(and/or alveolar epithelial cells) appeared to catabolize more
proteins. Alveolar macrophages have been demonstrated to be
important for the clearance of SP-A in rabbit lung (81). In
summary, macrophages may not play a major role in the
short-term removal of excess protein from the intact lung, but
they may be more important for long-term protein clearance or
for clearance of surfactant apoproteins. The role of alveolar
macrophages in the injured lung has not been studied yet.
Peptidases and proteases. Catabolism by the airway epithelial cells with absorption of smaller protein fragments was first
proposed as a mechanism for alveolar protein clearance by
Drinker and Hardenbergh (20). Various peptidases, such as
neutral endopeptidase and cathepsin H, are present on the
apical surface of the airway and alveolar epithelium and may
be important for clearing small peptides. (45, 64). Morimoto
and colleagues (62) suggested that aminopeptidases in the
apical cell plasma membranes of the alveolar epithelial cells
cultured on porous substrates could degrade a model dipeptide,
glycyl-L-phenylalanine. Protein might also be degraded after
endocytosis and transport to lysosomes in the epithelial cells.
Intra-alveolar degradation is not a major clearance mechanism
in the uninjured lung for most proteins, including serum
Invited Review
ALVEOLAR PROTEIN CLEARANCE
AJP-Lung Cell Mol Physiol • VOL
have been considered lately as possible routes of paracellular
transport. For example, edges of three epithelial cells frequently meet at one spot, a structure known as trijunctional a
complex (67). Trijunctional complexes leave a gap that contains glycocalyx but is still considerably larger than the opening of a tight junction (82). These gaps could serve to pass
protein. Defects left by removal of apoptotic cells have also
been recognized as potential though temporary pores for paracellular transport of macromolecules such as horseradish peroxidase (2).
Finally, studies are indicated to evaluate the role of receptormediated transcytosis of small quantities of protein in alveolar
physiology under normal conditions. Receptor-mediated removal of serum proteins could serve to maintain low protein
concentrations in the epithelial lining fluid under normal conditions, thus preserving low surface tension and optimal surfactant function (46). A carrier-mediated process could also be
involved in moving proteins, such as IgA, into the alveolar
space.
SUMMARY
The removal of protein from the distal air spaces of the lung
is an essential step in the recovery from pulmonary edema. A
variety of mechanisms have been studied for the removal of
alveolar protein, including transport by the mucociliary escalator, as well as alveolar degradation by macrophages. There is
some evidence for endocytosis across the alveolar epithelium,
a process known as transcytosis. There is also evidence for
paracellular diffusion through the distal epithelial barrier.
When protein concentrations in the distal air spaces are low (all
edema concentrations ⬍0.5 g/100 ml), protein transport demonstrates saturation kinetics, temperature dependence, and sensitivity to pharmacological agents that can stimulate or inhibit
endocytosis. In the presence of higher concentrations of alveolar protein, the rate of protein clearance appears to be proportional to concentration with no saturation kinetics. Protein
clearance is also inversely related to protein size and insensitive to endocytosis inhibition under these conditions. On the
basis of these findings, alveolar protein clearance can occur by
a receptor-mediated transcytosis mechanism at low protein
concentrations but probably proceeds by passive paracellular
mechanisms at higher concentrations, once the receptors are
saturated. Because protein concentrations in pulmonary edema
fluid are relatively high (⬎3 g/100 ml) (58), clearance of
alveolar protein probably occurs primarily by paracellular
pathways during the resolution of clinical pulmonary edema.
Transcytosis may be an important mechanism in regulating the
alveolar environment under nonpathological circumstances.
Also, alveolar degradation of protein may be an important
mechanism in long-term protein clearance, as well as in the
clearance of insoluble protein under pathological conditions
such as acute lung injury.
GRANTS
This work was supported by a Veterans Affairs Merit Review grant (R. H.
Hastings); National Institutes of Health Grants ES-09227, HL-51854, and
HL-51856; California Tobacco-Related Disease Research Program Grant
10RT-0161; and a grant from the Flight Attendants Medical Research Institute.
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Removal of the insoluble protein fragment will be much
slower than the removal of soluble protein components. Alveolar macrophages probably play a primary role in the removal
of insoluble proteins, matrix, and cell debris, although there is
very little quantitative information regarding this process. Observational studies in experimental conditions, such as bleomycin-induced lung injury (26), indicate that the process probably requires weeks for removal of insoluble protein in the
distal air spaces of the lung. The resolution process may also be
complicated by the presence of intra-alveolar fibrosis and
potentially by a loss of capillary blood flow and destruction of
some lung lymphatics.
With the availability of new therapies that reverse the
procoagulant environment and potentially stimulate fibrinolysis, it is possible that this clearance process of removal may be
accelerated. Conceivably, the use of activated protein C, for
example, might reverse the procoagulant environment, limit
deposition of insoluble protein in the air space in the lung, and
potentially activate fibrinolytic mechanisms that may lead to a
more rapid resolution of hyaline membranes and insoluble
protein complexes in the distal air spaces of the lung (43, 86).
Thus, although we have learned a great deal about the mechanisms for removal of the salt and water fraction of the edema
fluid from the distal air spaces of the lung (58), as well as the
mechanisms for removal of soluble protein (as discussed in this
review), much more information is required to understand the
intra-alveolar protein deposition that results in widespread
insoluble protein fragments deposited in the distal air spaces of
the lung.
Future directions. Protein transport has been studied extensively in normal lungs over relatively short periods, but pathological states, clearance of precipitated protein, and therapies
have received little attention. Future studies might explore
protein elimination in disease, clearance mechanisms for insoluble protein, and the impact of therapies on accelerating
liquid and/or protein clearance. For example, the previous
section mentions possible benefits of reversing the procoagulant state in the alveolar space. Other potential therapies
include proteins and peptides that open tight junctions for small
solutes and macromolecules (18, 44, 51). Such agents could be
investigated for their ability to speed alveolar protein clearance, although opening tight junctions could slow liquid clearance by allowing back flow into the air spaces. The impact of
beta agonists and other agents that speed liquid removal on
protein clearance is also an interesting question. Theoretically,
increased protein concentration could speed protein clearance
by augmenting the diffusion gradient or slow it by hastening
protein precipitation.
The site of paracellular protein passage along the bronchoalveolar tree under normal and pathological conditions is another
important area for exploration. The discovery that alveolar
fluid can undergo convective transport to distal upper airways
(83) lends credence to the hypothesis that tight junctions at the
bronchoalveolar interface could be involved in alveolar protein
clearance (see Fig. 4). Recent work has suggested that the
permeability of tight junctions is subject to physiological
regulation by factors such as peptides, secondary messengers,
kinases, and toxins (67). Thus paracellular protein clearance
could be a dynamic process that could respond to different
pathological situations and stimuli in an injury-specific manner. Types of channels other than junctions between two cells
L687
Invited Review
L688
ALVEOLAR PROTEIN CLEARANCE
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