Surfactant protein A (SP-A): the alveolus and beyond

KAVITA R. KHUBCHANDANI AND JEANNE M. SNYDER1

Department of Anatomy and Cell Biology, University of Iowa College of Medicine, Iowa City, Iowa
52242, USA
Surfactant protein A (SP-A) is the major
protein component of pulmonary surfactant, a material
secreted by the alveolar type II cell that reduces surface
tension at the alveolar airliquid interface. The function of SP-A in the alveolus is to facilitate the surface
tension-lowering properties of surfactant phospholipids, regulate surfactant phospholipid synthesis, secretion, and recycling, and counteract the inhibitory effects of plasma proteins released during lung injury on
surfactant function. It has also been shown that SP-A
modulates host response to microbes and particulates
at the level of the alveolus. More recently, several
investigators have reported that pulmonary surfactant
phospholipids and SP-A are present in nonalveolar
pulmonary sites as well as in other organs of the body.
We describe the structure and possible functions of
alveolar SP-A as well as the sites of extra-alveolar SP-A
expression and the possible functions of SP-A in these
sites.Khubchandani, K. R., Snyder, J. M. Surfactant
protein A (SP-A): the alveolus and beyond. FASEB J. 15,
59 69 (2001)
ABSTRACT

Key Words: lectins cystic fibrosis extra-alveolar sites innate immunity

PULMONARY SURFACTANT AND SP-A

Pulmonary surfactant is a lipoprotein synthesized
and secreted by alveolar type II cells that reduces the
surface tension at the lung alveolar airliquid interface
(1). Pulmonary surfactant is composed of phospholipids (80%), cholesterol (10%), and proteins
(10%). Four surfactant-associated proteins (SP) have
been identified and can be divided into two groups: the
hydrophilic proteins, SP-A and SP-D; and the hydrophobic proteins, SP-B and SP-C (1). SP-B and SP-C
greatly increase the adsorption of surfactant lipids onto
the surface film that lines the alveolus (1). SP-A is a
34 36 kDa protein that facilitates the surface tensionlowering properties of surfactant phospholipids in the
alveolus, regulates surfactant phospholipid synthesis,
secretion, and recycling, and counteracts the inhibitory
effects of plasma proteins released during lung injury
on surfactant function (2). SP-D is a 43 kDa protein that
has sequence homology to SP-A (3). Recent studies
suggest that both SP-A and SP-D are involved in innate
immune responses in the lung via their ability to bind
various pathogens including viruses, bacteria, fungi,
0892-6638/01/0015-0059/$02.25 FASEB

and particulates such as pollen grains and mite allergens (4).

The most abundant of the surfactant proteins is SP-A
(2). Human SP-A protein is encoded by two genes,
SP-A1 and SP-A2, which are 94% identical in nucleotide
sequence and 96% identical in amino acid sequence
(57). Each human SP-A gene gives rise to multiple
mRNA transcripts. The primary structure of SP-A protein consists of four domains: an amino-terminal domain, a collagenous domain, a neck domain, and a
carbohydrate recognition domain (CRD). In the lung
alveolus, human SP-A forms trimers that are thought to
be composed of two SP-A1 molecules and one SP-A2
molecules (8). As depicted in Fig. 1, the native SP-A
protein is made up of six SP-A trimers that form a
flower bouquet structure (8). The amino-terminal of
the SP-A molecule is a short peptide of 7 amino acids,
with a cysteine residue at position 6 that aids in the
formation of interchain disulfide bonds between SP-A
molecules. The collagenous domain of SP-A consists of
23 glycine-X-Y tripeptide repeats, with the Y most often
being a hydroxyproline (5). Cysteine residues involved
in SP-A trimer formation are also found in this region.
An interruption occurs after the 13th glycine-X-Y tripeptide repeat in SP-A, with a proline residue inserted
that introduces a flexible kink in the collagen region.
This kink causes the trimers to bend outward in different directions, giving the mature SP-A octadecamer a
flower bouquet appearance (8). The neck region of
SP-A consists of a short sequence of hydrophobic
residues and an amphipathic helix, whereas the CRD
contains a calcium-dependent, carbohydrate binding
site (9). Post-translational modifications of SP-A include cleavage of a signal peptide, hydroxylation of
proline residues, sulfation, acetylation, and glycosylation with N-linked oligosaccharides (2).
SP-A is a member of the collectin family of C-type
lectins, which includes mannose binding protein, the
C1q component of complement, the bovine protein
conglutinin, and surfactant protein D. The human SP-A
genes, along with the genes for the other collectins, are
found together on chromosome 10 (5). Collectins are
characterized by a collagen-like domain and the ability
to bind carbohydrates. They bind to specific carbohy1
Correspondence: Department of Anatomy and Cell Biology, 1550 Bowen Science Bldg., University of Iowa College of
Medicine, Iowa City, IA 52242, USA. E-mail: jeannesnyder@uiowa.edu

59

Figure 1. Structural models of SP-A protein. Left: The SP-A

trimer. The collagen-like regions of three SP-A monomers
form a triple helix rod-like region whereas the globular head
of the trimer is formed by the lectin domains of the three
SP-A monomers. It has been proposed that the human SP-A
trimer is a heterotrimer consisting of two SP-A1 molecules
(black) and one SP-A2 molecule (red). Right: The SP-A
octadecamer. Six SP-A trimers associate to form a flower
bouquet-like structure.

drates on the surface of bacterial and viral pathogens

and act as an opsonin (4). They also mimic C1q in the
activation of the classical complement pathway and can
activate macrophages and other phagocytic cells (4).
Mutations in one of the collectins, mannose binding
protein, have been associated with increased susceptibility to infectious disease (10). To date, no diseases
have been associated with genetic mutations in human
SP-A, although certain SP-A alleles may be associated
with an increased risk of developing respiratory distress
syndrome (RDS) (11). Transgenic mice with SP-A gene
deletion have no major abnormalities in lung respiratory function but are more susceptible to infection with
group B Streptococcus, Pseudomonas aeruginosa, respiratory syncytial virus, and Mycoplasma pulmonis (1215).
Recently, Harrod and colleagues observed that exogenous human SP-A enhanced viral clearance and inhibited inflammation in SP-A gene-deleted mice infected
with recombinant adenovirus (16).

SP-A RECEPTORS
SP-A acts as an opsonin by recognizing and binding to
carbohydrates on the surface of microorganisms. SP-A
isolated from rat, dog, or human binds to macrophages, stimulates their oxidative activity, and promotes lymphocyte proliferation (17, 18). Human SP-A
increases the production of proinflammatory cytokines
(TNF-, interleukins) in rat alveolar macrophages and
peripheral blood mononuclear cells (19). These effects
as well as the increase in the expression of cell surface
proteinsCD14, CD54, and CD11b have been observed in a human monocytic/macrophage cell line,
THP-1 cells (20).
Several proteins have been suggested as a candidate
for the cellular SP-A receptor. A 126 kDa protein
named C1qRp (i.e., C1q receptor that mediates phago60

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January 2001

cytosis) has been described that binds C1q, mannose

binding lectin, and SP-A (21). Another SP-A receptor,
identified by affinity chromatography, is a 210 kDa
protein present in rat lung and the U937 cell line (22).
Binding of SP-A to this receptor is calcium dependent
and based on proteinprotein interactions, not carbohydrate recognition, since the binding of SP-A to this
protein is not inhibited by mannan or other sugars.
Polyclonal antibodies against this SP-A receptor block
the inhibitory effects of SP-A on phospholipid secretion
by alveolar type II cells and block SP-A-mediated uptake
of bacillus Calmette-Guerin by rat macrophages (23). A
third SP-A receptor identified on rat alveolar type II
cells is a 200 kDa protein that may be responsible for
SP-A-mediated uptake of phosphatidylcholine by alveolar type II cells (24).

REGULATION OF SP-A
SP-A is undetectable in human fetal lung tissue during
the early part of the second trimester, i.e., prior to the
differentiation of the alveolar epithelium (25). Differentiated type II cells that contain lamellar bodies are
observed in human fetal lung by 22 wk of gestation,
and active surfactant secretion into amniotic fluid
occurs after 30 wk of gestation. Immunoreactive SP-A
can be detected in amniotic fluid at 30 to 32 wk of
gestation (26). Khoor et al. have detected immunoreactive SP-A in prealveolar-type II cells at 20 wk gestation
(27).
SP-A gene expression is regulated by several growth
factors, hormones, and regulatory agents (2). SP-A
levels are increased by treatment with cAMP analogs
(28, 29), epidermal growth factor (30), interferon
gamma (31), prostaglandins (32), oxygen (33, 34),
endotoxin (35, 36), and -adrenergic agonists (37).
SP-A is decreased by insulin (38), transforming growth
factor (TGF-) (30), tumor necrosis factor (TNF-)
(39), and 12-O-tetradecanoyl-phorbol-13-acetate (40).
Both inhibitory and stimulatory effects of glucocorticoids on SP-A expression have been reported, with the
inhibitory effect observed at higher hormone concentrations (41, 42).

FUNCTIONS OF SP-A
Surface tension
Once lamellar bodies are secreted from the alveolar
type II cell, they assume a geometric lattice-like morphology known as tubular myelin (1). Tubular myelin is
thought to retain surfactant phospholipids in the alveolus until they spread into the monolayer that lines the
alveolus. SP-A is localized in the corners of this lattice
structure and is required for the formation of tubular
myelin (43). Infants suffering from RDS have a deficiency of both tubular myelin and SP-A (44), consistent
with a role for SP-A in the formation of tubular myelin.

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KHUBCHANDANI AND SNYDER

In agreement with these observations, tubular myelin is

reduced in SP-A gene-deleted mice (45). SP-A enhances the rate of formation of a phospholipid surface
film at an airliquid interface in vitro (46), probably by
binding to dipalmitoylphosphatidylcholine, the most
abundant phospholipid in surfactant (47). SP-A binds
to a receptor on type II cells and enhances the uptake
of surfactant phospholipids for recycling. Surfactant
clearance is also carried out by macrophages and this
function is likewise enhanced by SP-A (48). SP-A has
also been shown to inhibit the secretion of surfactant
phospholipids by rat alveolar type II cells in vitro, data
suggestive that SP-A is involved in a negative feedback
loop that regulates surfactant homeostasis in the alveolus (49). Surprisingly, pulmonary function is not altered in SP-A gene-deleted mice (45). However, it is still
possible that SP-A may play a role in surfactant function
when surfactant phospholipid levels are lowfor example, early in development.
SP-A prevents inhibition of surfactant activity by
blood proteins
Leakage of blood components into the alveolar space as
a result of lung injury has been implicated in the
pathology of respiratory distress syndrome (50). Plasma
proteins have been shown to inhibit surfactant activity
both in vitro and in vivo (50, 51). Bovine SP-A reduces
the inhibition of surfactant activity mediated by plasma
proteins (52). This is specific to SP-A since the addition
of bovine serum albumin to surfactant did not produce
the same effect. The inhibition of surfactant activity is
abolished only when SP-A is added to the surfactant
preparation before the addition of the inhibitory
plasma proteins. The addition of lysophosphatidylcholine (lyso-PC) to the surfactant preparations also inhibits surfactant activity; however, the addition of SP-A has
no effect on the lyso-PC-mediated inhibition (52),
suggesting that the effect of SP-A in inhibiting the
surface tension reduction is specific to plasma proteins.
Consistent with these observations, surfactant from
SP-A knockout mice is more sensitive to inhibition by
plasma proteins than is surfactant obtained from wildtype mice (53).
SP-A binds LPS
Lipopolysaccharide (LPS), or endotoxin, is a major
component in the cell walls of gram-negative bacteria.
LPS consists of lipid A, which is its biologically active
component, a core carbohydrate region, and a terminal
polysaccharide of variable length and composition, i.e.,
the O-antigen domain (54). Lipid A and the core
region make up rough LPS, whereas smooth LPS
consists of the core region and the O-antigen domain.
Human SP-A binds rough LPS via lipid A, and this
binding is not inhibited by either the addition of
carbohydrates or the removal of N-linked sugars (54).
SP-A does not bind smooth LPS. LPS participates in
many events associated with the inflammatory response
SP-A: BEYOND THE LUNG ALVEOLUS

at the alveolar level, and has been shown to increase the

production of colony-stimulating factor (CSF) by cultured alveolar type II cells and macrophages (55).
Human and rat SP-A, when added alone, also cause an
increase in CSF secretion by cultured rat alveolar type II
cells and macrophages (55). However, this increase is
reversed when the SP-A and LPS are added together
(55). Human SP-A enhances the binding of LPS to
alveolar macrophages, but inhibits the binding of LPS
to neutrophils (56). Human SP-A binds to gram-negative bacteria via LPS and facilitates their aggregation,
phagocytosis, and killing by rat macrophages (57). The
interactions of SP-A and LPS suggest that SP-A may
influence the pathogenicity of gram-negative bacteria
in the pulmonary alveolus.
Activation of macrophages
The killing of microorganisms by macrophage phagocytosis can be initiated by a variety of mechanisms,
including the secretion of proteolytic enzymes and the
production of toxic oxygen metabolites, which together
constitute the respiratory burst. Rat, canine, and human SP-A can cause the respiratory burst in rat and
human macrophages and also enhance the chemotaxis
of activated macrophages (58, 59). Tenner (60) showed
that immobilized human SP-A binds to human monocytes and up-regulates complement receptor and immunoglobulin receptor-mediated phagocytosis. Human SP-A also up-regulates the human macrophage
mannose receptor and enhances phagocytosis (61, 62).
Aggregation of SP-A may also lead to the activation of
macrophages and the stimulation of phagocytosis (57).
Rat, canine, and human SP-A stimulates the generation
of oxidative activity (17) and promotes lymphocyte
proliferation in rat macrophages (18). Human SP-A
also promotes the production of proinflammatory cytokines such as TNF- and interleukins in the THP-1
cell line, a monocytic cell line that can be stimulated in
vitro to differentiate into macrophages (63). Human
and rat SP-A have also been shown to increase nitric
oxide production in rat alveolar macrophages (64).
Human SP-A activates macrophages via a phosphoinositide/calcium signaling pathway (65). Surfactant lipids
inhibit some macrophage functions, including several
that are stimulated by SP-A (18, 20).
Human SP-A has been shown to increase neutrophil
uptake and killing of lung pathogens (66). In response
to airway infection, cytokines and other mediators of
inflammation released by phagocytes and by the airway
epithelium can lead to a chronic inflammatory response in the airways (18, 67). SP-A suppresses these
inflammatory responses (68). In agreement with this
concept, it has been reported that the concentrations
of proinflammatory cytokines are greater in SP-A genedeleted mice infected with P. aeruginosa than in wildtype mice (13). Other investigators have reported that
a heightened immune response is associated with a
decrease in the ratio of SP-A to surfactant phospholipid
in cystic fibrosis patients (69).
61

SP-A acts as an opsonin for pathogens

SP-A is thought to bind to lung pathogens via its CRD,
and in this way promote binding and phagocytosis of
pathogens by macrophages (70). The efficiency of
interaction between SP-A and microorganisms may
depend on the oligomeric state of the SP-A protein,
which may vary in different disease states. There appears to be a shift toward increased abundance of lower
oligomeric forms of SP-A in several disease states (71).
Table 1 summarizes the microorganisms that have been
shown to bind SP-A and whether SP-A enhances their
phagocytosis by macrophages.
Human SP-A binds to Aspergillus fumigatus (72), a
fungus that causes pulmonary aspergillosis, and Candida albicans (73), and increases their uptake by human
macrophages. Other fungi that human SP-A interacts
with include acapsular Cryptococci neoformans (74) and
Pneumocystis carinii (73).
SP-A binds to and stimulates the phagocytosis of both
gram-positive and gram-negative bacteria. Human SP-A
stimulates the macrophage uptake of gram-positive
bacteria such as Staphylococcus aureus, S. pneumoniae, and
group A Streptococcus (58, 70, 75). All these organisms
have been shown to cause airway infections. Human
SP-A increases the phagocytosis and killing of certain
strains of gram-negative bacteria such as Esherichia coli
that express rough LPS (54, 57), as well as Haemophilus
influenzae type A (70) and Klebsiella pneumoniae (76).
Binding of SP-A to P. aeruginosa is controversial; however, human SP-A has been shown to enhance its
phagocytosis by rat alveolar macrophages (62). Human
SP-A binds to M. pulmonis, a pathogen that causes
pneumonia in mice, and increases the phagocytosis of
this pathogen by human alveolar macrophages (15).
SP-A isolated from human alveolar proteinosis lavage
and recombinant rat SP-A have both been shown to
enhance the binding and phagocytosis of Mycobacterium
tuberculosis by human macrophages (61) and to increase

the binding and phagocytosis of bacillus CalmetteGuerin by rat macrophages and human monocytes via
the 210 kDa SP-A receptor (23). Human SP-A also binds
the herpes simplex and influenza A viruses via its
carbohydrate moiety and enhances their phagocytosis
by human and rat macrophages (77, 78).
Other mechanisms of SP-A action in local defense
may exist. For example, human SP-A has been shown to
cause aggregation of pathogens, thus facilitating their
clearance by the mucociliary escalator (66). Alternatively, the binding of human SP-A to some pathogens
may inhibit their binding to the airway epithelium (79).
SP-A levels in disease
Abnormalities in SP-A levels have been detected in
several disease states. For example, SP-A levels are
decreased in the amniotic fluid of diabetic pregnant
mothers (80, 81). Pregnancies characterized by low
levels of amniotic fluid SP-A before delivery are associated with an increased risk of the infant being born
with RDS (82). In agreement with this observation,
SP-A levels are low in tracheal aspirates obtained from
infants with RDS (44). Respiratory dysfunction in animal models of lung injury is correlated with decreased
amounts of large surfactant aggregates, forms of surfactant that are enriched in SP-A (83). A decrease in SP-A
levels has also been observed in the bronchoalveolar
lavage (BAL) of adult patients with acute RDS (ARDS)
(84).
BAL SP-A levels in patients with cystic fibrosis (CF)
and lower airway infections are higher than in CF
patients without infection (85). This increase may
occur in response to infection. However, a decrease in
BAL SP-A levels was observed in patients with bacterial
and viral pneumonia (86, 87). Decreased BAL SP-A
levels have also been reported in bronchopulmonary
dysplasia with infection in baboons (88) and in inter-

TABLE 1. Pathogens that bind SP-A

Organism

Group A Streptococcus
Mycoplasma pulmonis
Staphylococcus aureus
Streptococcus pneumoniae
Pseudomonas aeruginosa
Escherichia coli
Klebsiella pneumoniae
Haemophilus influenzae
Aspergillus fumigatus
Cryptococci neoformans
Pneumocystis carinii
Mycobacterium tuberculosis
Bacillus Calmette Guerin
Influenza A
Adenovirus
Herpes simplex

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January 2001

SP-A increases
phagocytosis by
macrophages

Source
SP-A

Macrophages

Reference

Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
No
No
Yes
Yes
Yes
Yes
Yes

Human
Human
Human
Human
Human
Human
Human
Human and rat
Human
Human
Human
Human
Human
Human
Human
Human

Rat
Mouse
Rat
Rat
Rat
Rat
Rat and guinea pig
Rat
Human
Human
Human
Human
Rat
Human
Mouse
Rat

70
15
58
70
118
57
76
70
72
117
73
61
23
79
16
78

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KHUBCHANDANI AND SNYDER

stitial pneumonia with collagen vascular disease (84).

On the other hand, BAL SP-A and serum SP-A levels in
patients with AIDS-related pneumonia are increased
when compared to normal healthy subjects (89). This
increase is not pathogen specific and is seen in infections with P. carinii and non-P. carinii infections.
Whether the increase in SP-A in AIDS-related pneumonia is a cause or an effect of infection is unclear.
Decreased SP-A levels are observed in lavage of patients
with idiopathic pulmonary fibrosis (IPF) (84, 90). However, another report showed no difference in SP-A
levels in BAL of IPF patients when compared to controls. SP-A levels are increased in BAL of patients with
pulmonary alveolar proteinosis (PAP), sarcoidosis, and
hypersensitivity pneumonitis (9193). PAP is frequently
associated with mycobacterial infections, a finding that
supports the concept that SP-A may facilitate the binding and entry of bacteria into cells lining the respiratory
tract.
Since it is known that blood proteins may leak into
the alveolar spaces as a result of lung injury, it is
reasonable to assume that surfactant proteins may leak
into the vascular spaces in disease. Serum SP-A levels
may be useful as an indicator of lung function and
alveolar-capillary membrane injury due to disease. Kuroki et al. (94) reported an increase in serum SP-A
levels in patients with idiopathic pulmonary fibrosis,
pulmonary alveolar proteinosis, tuberculosis, bacterial
pneumonia, and chronic pulmonary emphysema (84).
They observed no difference in serum SP-A levels in
patients with bronchial asthma and sarcoidosis when
compared to healthy individuals (84). Serum levels of
SP-A are also increased in patients with acute cardiogenic pulmonary edema and in ARDS (94).

SP-A IN NONALVEOLAR SITES IN THE

RESPIRATORY SYSTEM
SP-A in the conducting airways
Tracheal surfactant from porcine lungs has the same
phospholipid composition as BAL surfactant, which
suggests an alveolar origin for this surfactant (95).
However, SP-A mRNA and protein have been detected
in human fetal tracheal and bronchial epithelium and
submucosal glands (27, 96, 97). Our laboratory (97, 98)
and that of Saitoh et al. (99) have demonstrated that
only the SP-A2 gene is expressed in human fetal and
adult trachea and bronchi. SP-A2 is also the predominant SP-A protein present in the adult human trachea
(98, 99). We have localized SP-A2 mRNA to the serous
cells of submucosal glands of the adult human conducting airways by in situ hybridization (Fig. 2; 98). The
SP-A2 protein present in the fetal and adult tracheal
submucosal glands has the same molecular weight and
post-translational modifications as that of distal lung
heterotrimeric SP-A protein (Fig. 3; 98). No studies to
evaluate the regulation or function of the conducting
airway form of SP-A protein have been performed to
date. We hypothesize that this novel SP-A protein
functions in combination with other submucosal gland
serous cell secretions in local anti-microbial host defense mechanisms in the airways. There is no gene

SUMMARY OF SP-A FUNCTION IN THE

ALVEOLUS
Surfactant protein A has been shown to regulate the
homeostasis of surfactant phospholipids and facilitate
the lowering of surface tension in the alveolus. SP-A
also prevents the inhibition of surfactant function by
plasma proteins that have leaked into the injured
alveolus. Recent studies using SP-A knockout mice have
revealed that no major abnormalities in normal lung
function exist in these animals. However, the SP-A
gene-deleted mice are more susceptible to infection
with group B Streptococcus pneumonia, P. aeruginosa, M.
pulmonalis, and respiratory syncytial virus. In addition,
surfactant function is inhibited by plasma proteins in
SP-A gene-deleted mice to a greater degree than in
wild-type mice. Several in vivo and in vitro studies have
shown that SP-A binds to bacteria and/or macrophages
and also enhances the phagocytosis and killing of lung
pathogens. Thus, the major role of SP-A in the alveolus
may be related to local host defense mechanisms.
SP-A: BEYOND THE LUNG ALVEOLUS

Figure 2. In situ hybridization of SP-A mRNA in adult human

lung bronchi. A, C, E) Bright field images; B, D, F) corresponding dark field images. SP-A mRNA was not detected in
the surface epithelium (asterisks) or in the ciliated duct (CD)
portion of the submucosal glands. The lumen of the airway is
indicated by an L. SP-A mRNA was present in some tubules
of the submucosal glands (arrows) but absent in others (T).
The bar at the lower right corner indicates 100 m (from
Khubchandani et al., ref 98).
63

Figure 3. Immunoblot analysis of SP-A protein in adult

human tracheal submucosal tissue. Total protein homogenates were separated by polyacrylamide gel electrophoresis
and transferred to a membrane, then probed using antihuman SP-A antibodies and enhanced chemiluminescence
(ECL). A) Human SP-A purified from alveolar proteinosis
material was used as a positive control (lane 1: lower arrow
depicts the 35 kDa SP-A monomer; upper arrow depicts the
65 kDa SP-A dimer). A 35 kDa SP-A immunoreactive band
was detected in cultured human fetal lung explants (lane 2),
as well as in adult human tracheal submucosal tissues (lanes
3 6). The higher molecular weight band in lanes 3 6 may
represent an unglycosylated dimer of SP-A. The amount of
SP-A protein present in the tracheal samples varied considerably. B) Peptide-N glycosidase F (PNGF) digestion of the SP-A
protein in adult human tracheal submucosal tissues to remove N-linked carbohydrates. SP-A isolated from human
alveolar proteinosis material and protein homogenates from
adult human tracheal submucosal tissues were digested with
PNGF and analyzed by immunoblotting. Intact SP-A immunoreactive bands (35 kDa monomer, 65 kDa dimer, long
arrows) were detected in the undigested purified SP-A (lane
1) and in undigested adult human tracheal submucosal
tissues (lanes 3, 5, 7). After digestion with PNGF, lower
molecular mass bands (31 kDa monomer, 50 kDa dimer,
short arrows) were detected in the purified human SP-A (lane
2) and in the adult human tracheal submucosal tissues (lanes
4, 6, 8) (from Khubchandani et al., ref 98)

tubes were irrigated with saline, presumably because

the washing removed the surfactant (100). In another study, natural surfactant from guinea pig lungs,
saline, or a mixture of phospholipids was injected
into the middle ears of rats (101). The pressure
required to open the Eustachian tube decreased
significantly in the surfactant and phospholipidtreated animals, and natural surfactant also reduced
the pressure more than the phospholipid mixture
(101). Western blot analysis of middle ear effusion
fluid revealed an 80 kDa immunoreactive protein
that was recognized by monoclonal antibodies directed against human SP-A (102). In another study,
Yamanaka et al. (103) detected SP-A immunoreactivity in middle ear effusions obtained from patients
with otitis media with effusion. Our laboratory has
demonstrated the presence of SP-A mRNA and protein in the middle ear epithelium and in submucosal
glands in the paranasal sinuses of adult rabbits (ref
104; Fig. 4 and Fig. 5). We also observed higher levels
of SP-A mRNA in infected rabbits when compared to
pathogen-free rabbits. Recently, Paananen and coworkers detected the presence of SP-A in porcine
Eustachian tube epithelium (105). There are no
reports as yet concerning the function of SP-A in the
middle ear or paranasal sinuses sites. Surfactant
phospholipids may function in reducing the surface
tension in the Eustachian tube and in this manner
prevent its collapse. Collapse of the Eustachian tube
may hinder ventilation of the middle ear and increase the risk of infection (106). We and others have
also speculated that SP-A may contribute to local
immunity in the middle ear, Eustachian tube and
paranasal sinuses.

deletion model available for studying the airway SP-A

protein, because the mouse does not have submucosal
glands in its conducting airways and has only a few
glands located at the top of the trachea.
SP-A in the Eustachian tube, middle ear, and
paranasal sinuses
Immunoreactive SP-A has been detected in the cells
lining the Eustachian tube in the human. The presence of a surface tension-lowering substance in the
Eustachian tube had previously been postulated
when it was demonstrated that the opening of guinea
pig Eustachian tubes became more difficult after the
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January 2001

Figure 4. SP-A mRNA in rabbit middle ear and sinus tissues.

Total RNA was isolated, separated by gel electrophoresis,
transferred to a membrane, and probed with a radiolabeled
rabbit SP-A cDNA. A) Northern blot analysis of SP-A mRNA
(arrow) in adult rabbit lung (lane A), trachea (lane B), and
maxillary sinus (lane C). B) Northern blot analysis of SP-A
mRNA (arrow) in rabbit lung (lane A), and middle ear (lane
B) (from Dutton et al., ref 104)

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be involved in regulating surfactant phospholipid uptake and secretion by the gastrointestinal tract epithelium. SP-A in these sites may also aid in host defense
functions via presentation of pathogens to macrophages and/or by prevention of attachment of pathogens to the epithelium.
SP-A in the prostate gland

Figure 5. SP-A immunolocalization in lung, middle ear, and

maxillary sinus tissues. A) SP-A was present in alveolar type II
cells (arrows) of adult rabbit lung tissue. B) SP-A was localized
in the surface epithelium (arrows) of the rabbit middle ear
tissues. C) SP-A was localized in submucosal glands (arrows) of
maxillary sinus tissues. D) No staining was observed in maxillary sinus tissues when PBS was used instead of primary
antibody. Arrows denote brown staining for SP-A. SM indicates submucosal tissue, L indicates lumen. The bar at the
lower right corner indicates 100 m. (from Dutton et al., ref
104)

SP-A IN NONRESPIRATORY TRACT SITES

SP-A in the gastrointestinal tract
Hills et al. detected a pulmonary surfactant-like material in the gastrointestinal mucosa as early as 1983
(107). A hydrophobic layer of surface-active phospholipids was detected between the apical border of epithelial cells of the gastrointestinal tract and the lumenal
contents, and was designated gastrointestinal surfactant
(107, 108). In the stomach, surfactant phospholipids
were detected in multilamellar structures on the mucosal surface (109). Epithelial cells in the intestine of the
rat have been shown to produce SP-A mRNA and
protein (109). The SP-A protein made in the intestine
has the same charge and molecular weight heterogeneity as the SP-A protein found in the lungs (109).
Moreover, the intestinal SP-A protein was found only in
epithelial cells of the jejunal and colonic mucosa, and
was not present in the gastric mucosa (108). Lu (110)
detected no SP-A mRNA in human small intestine and
colon by Northern blot analysis. More recently, however, Eliakim and co-workers have reported the presence of surfactant-like particles and SP-A in both rat
and human colon (111). The lamellar bodies in these
sites contain saturated phosphatidylcholine and are
able to lower surface tension. It has been proposed that
gastrointestinal surfactant may be involved in lubrication within the gastrointestinal tract and in reducing
surface tension in the intestine, where peristalsis may
be benefited. Surfactant may also act as a barrier
against ulcer-causing agents. The specific function of
SP-A in the gastrointestinal tract has not been determined, but several authors have speculated that it may
SP-A: BEYOND THE LUNG ALVEOLUS

Northern blot analysis revealed the presence of SP-A

mRNA in the human prostate (110). Our laboratory
has detected SP-A mRNA in human prostate by Northern blot analysis using a human SP-A cDNA probe (Fig.
6). We also detected an 35 kDa SP-A immunoreactive
protein in human seminal fluid (Fig. 6). To our knowledge, these are the only reports concerning the presence of SP-A mRNA or protein in prostate. Since there
are no published reports concerning the presence of a
surfactant-like material in the prostate, we can only
speculate that the SP-A present in the prostate and
prostatic secretions may be involved in host defense
mechanisms in the male and perhaps also in the female
reproductive system. These organs communicate with
the outside environment and are susceptible to pathogen exposure and subsequent infection. SP-A in these
sites may act along with proteins such as seminal
plasmin, an anti-microbial protein present in bovine
seminal plasma that prevents invasion of infectious
pathogens (112).
SP-A in the thymus and spleen
Alcorn et al. created transgenic mice with fusion genes
composed of the 5 flanking regions of the rabbit SP-A
gene linked to a human growth hormone (hGH) reporter
gene (113). In addition to detecting high levels of hGH

Figure 6. Presence of SP-A mRNA and protein in human

prostate (A, lane 2) and human seminal fluid (B, lane 2),
respectively. A) Northern blot analysis of SP-A mRNA. 2 g of
mRNA from human lung tissue and 2 g of mRNA from
human prostate was separated by gel electrophoresis, transferred to a membrane, and probed with a radiolabeled
human SP-A cDNA. SP-A mRNA was detected in human lung
tissue (lane 1) and human prostate (lane 2). B) Immunoblot
analysis revealed the presence of SP-A protein in purified
human alveolar proteinosis material (50 ng, lane 1) and in
human seminal fluid (100 g, lane 2).
65

mRNA in the lung, reporter gene expression was also

frequently detected in the thymus and spleen. One other
investigator has described low levels of SP-A mRNA in the
human thymus (110). The role of SP-A in the spleen and
thymus is unlikely related to surface tension but may be
related to host defense function.

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SP-A in mesothelium and synovium

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