Am J Physiol Lung Cell Mol Physiol
281: L517–L523, 2001.
EB2001 symposium report
Lung surfactant and reactive oxygen-nitrogen species:
antimicrobial activity and host-pathogen interactions
Downloaded from http://ajplung.physiology.org/ by 10.220.33.6 on June 18, 2017
JUDY M. HICKMAN-DAVIS,1 FERRIC C. FANG,2 CARL NATHAN,3
VIRGINIA L. SHEPHERD,4 DENNIS R. VOELKER,5 AND JO RAE WRIGHT6
1
Department of Anesthesiology, University of Alabama at Birmingham, Birmingham,
Alabama 35249; 2Clinical Microbiology Laboratory, University of Colorado Health
Sciences Center, Denver 80262; 5Department of Medicine, National Jewish Center
for Immunology and Respiratory Medicine, Denver, Colorado 80206; 3Department
of Microbiology and Immunology, Weill Medical College of Cornell University,
New York, New York 10021; 4Department of Medicine, Vanderbilt University
School of Medicine, Nashville, Tennessee 37212; and 6Department
of Cell Biology, Duke University, Durham, North Carolina 27710
Hickman-Davis, Judy M., Ferric C. Fang, Carl Nathan, Virginia
L. Shepherd, Dennis R. Voelker, and Jo Rae Wright. Lung surfactant and reactive oxygen-nitrogen species: antimicrobial activity and
host-pathogen interactions. Am J Physiol Lung Cell Mol Physiol 281:
L517–L523, 2001.—Surfactant protein (SP) A and SP-D are members of
the collectin superfamily. They are widely distributed within the lung,
are capable of antigen recognition, and can discern self versus nonself.
SPs recognize bacteria, fungi, and viruses by binding mannose and
N-acetylglucosamine residues on microbial cell walls. SP-A has been
shown to stimulate the respiratory burst as well as nitric oxide synthase
expression by alveolar macrophages. Although nitric oxide (NO䡠) is a
well-recognized microbicidal product of macrophages, the mechanism(s)
by which NO䡠 contributes to host defense remains undefined. The purpose of this symposium was to present current research pertaining to the
specific role of SPs and reactive oxygen-nitrogen species in innate immunity. The symposium focused on the mechanisms of NO䡠-mediated
toxicity for bacterial, human, and animal models of SP-A- and NO䡠mediated pathogen killing, microbial defense mechanisms against reactive oxygen-nitrogen species, specific examples and signaling pathways
involved in the SP-A-mediated killing of pulmonary pathogens, the
structure and binding of SP-A and SP-D to bacterial targets, and the
immunoregulatory functions of SP-A.
collectins; surfactant protein A; nitric oxide; peroxynitrite; innate immunity; macrophages; signal transduction
THE RESPIRATORY TRACT IS EXPOSED to a multitude of toxic
and infectious agents on a regular basis, and yet, in the
healthy lung, disease is a relatively rare event. Innate
immune mechanisms in the lung include ciliary (mechanical) clearance, a cough reflex (also mechanical),
Address for reprint requests and other correspondence: J. Hickman-Davis, Dept. of Anesthesiology, Univ. of Alabama at Birmingham, 619 South 19th St., THT 940, Birmingham, AL 35294 (E-mail:
[email protected]).
http://www.ajplung.org
and cellular defenses. Within the alveolar lining fluid,
surfactant proteins (SPs) provide a first line of defense
against invading organisms before specific immunity
to them can develop. SP-A and SP-D are members of a
family of proteins known as C-type lectins or collectins
(collagen-like lectin) (16) that are important in innate
immunity. These proteins recognize bacteria, fungi,
and viruses by binding mannose and N-acetylglucosamine residues on microbial cell walls. SP-A has
been shown to stimulate the respiratory burst as well
as nitric oxide synthase expression by alveolar macro-
1040-0605/01 $5.00 Copyright © 2001 the American Physiological Society
L517
L518
EB2001 SYMPOSIUM REPORT
NITRIC OXIDE-MEDIATED MECHANISMS OF
SALMONELLA KILLING BY MACROPHAGES1
Nitrogen oxides produced by phagocytic cells have
been strongly implicated in the antimicrobial defense
against parasites, fungi, bacteria, and viruses (13).
Both humans and experimental animals produce dramatically increased quantities of NO䡠 during infection,
and expression of nitric oxide synthase can be directly
demonstrated in infected tissues. Inhibition of nitric
oxide synthase exacerbates microbial proliferation during experimental infections or phagocyte-killing assays, and a variety of chemical NO䡠 donors have been
shown to inhibit or kill diverse microbial species in
vitro (13). Although the antimicrobial activity of NO䡠
can be shown to be synergistic with that of reactive
oxygen species, observations in Salmonella-infected
murine peritoneal macrophages indicate that initial
1
bacterial killing by phagocytes is primarily dependent
on phox, with subsequent sustained inhibition of bacterial growth being mediated by NO䡠 (47, 53). Similarly, in a murine systemic Salmonella infection model,
phox appears to be essential for an initial reduction in
organism burden, whereas NO䡠 plays a later role in the
inhibition of replication of residual bacteria (30, 47).
This sequential expression of oxidative and nitrosative
antimicrobial mechanisms may maximize initial microbial killing while limiting collateral damage to the
host (52).
Microbes can employ a variety of defensive strategies
against nitrogen oxides, including avoidance, expression of stress regulons, direct or indirect detoxification,
radical scavenging, repair of nitrosative damage, and
inhibition of production. Recent studies suggest that
microbial DNA replication is a particularly critical
target of reactive nitrogen and oxygen species. An
improved understanding of NO䡠 targets in bacteria and
relevant mechanisms of bacterial resistance to NO䡠 will
shed new light on microbial pathogenesis and lead to
the discovery and application of new therapies for
infectious diseases.
KILLING OF PATHOGENS BY HUMAN AND MURINE
ALVEOLAR MACROPHAGES: INVOLVEMENT OF
REACTIVE OXYGEN-NITROGEN INTERMEDIATES2
The importance of NO䡠 for the clearance of pulmonary bacterial pathogens, including Mycobacterium tuberculosis (34) and Mycoplasma pulmonis, has been
characterized in a number of animal model systems in
vivo and in vitro (17). Extrapolation of the findings
from these animal model systems to the human condition allows valuable insight into disease pathogenesis;
however, it has long been recognized that NO䡠 production is differentially regulated in different species (20).
The involvement of NO䡠 in the pathogen clearance in
human diseases has been indirectly inferred from malaria studies, where increased levels of nitrate and
nitrite (the stable breakdown products of NO䡠) have
been correlated with improved prognosis (1), from
studies of tuberculosis outbreaks in pediatric populations (50), and from a number of other clinical studies
(reviewed in Ref. 23). In vitro attempts to stimulate
production of NO䡠 by human monocytes have failed,
and the study of NO䡠 production by human AMs has
been confined to systems in which these cells are already stimulated, such as idiopathic pulmonary fibrosis (36) and cancer (12).
The ability of the pulmonary collectin SP-A to modulate NO䡠 production by AMs has been demonstrated
with transformed cell lines (21) and primary rat AMs
(2). The importance of SP-A and NO䡠 for pulmonary
bacterial clearance was shown in a mouse model of
respiratory mycoplasmosis (17). In vitro mouse AMs
infected with M. pulmonis produce significant amounts
of NO䡠 but do not kill mycoplasmas; however, the
addition of SP-A to these cells increases NO䡠 produc2
Presented by Ferric Fang.
AJP-Lung Cell Mol Physiol • VOL
Presented by Judy M. Hickman-Davis.
281 • SEPTEMBER 2001 •
www.ajplung.org
Downloaded from http://ajplung.physiology.org/ by 10.220.33.6 on June 18, 2017
phages (AMs). Superoxide, produced by the membranebound NADPH phagocyte oxidase (phox) of macrophages, combines with nitric oxide (NO䡠) to form the
strong oxidant and bactericidal compound peroxynitrite (ONOO⫺) at near the diffusion limit for these two
molecules.
Although NO䡠 is a well-recognized microbicidal molecule of macrophages, the mechanism(s) by which NO䡠
contributes to host defense remains undefined. NO䡠
may have a direct microbicidal effect through 1) a
reaction with iron or thiol groups on proteins, forming
iron-nitrosyl complexes that inactivate enzymes important in DNA replication or mitochondrial respiration;
2) formation of such reactive oxidant species as
ONOO⫺; 3) synergism with the hydroxyl radical to
induce double-strand DNA breakage; or 4) inhibition of
antioxidant metalloenzymes such as catalase, thereby
increasing hydrogen peroxide and hydroxyl concentrations (10).
In general, the understanding of pulmonary host
defense mechanisms lags behind that of other systems
because of the poor accessibility and complexity of the
lung environment. If the innate mechanisms of the
lung are of primary importance in the defense against
bacterial infections, a better understanding of the role
of SPs and reactive oxygen and nitrogen species in the
early immune response may allow for the development
of novel therapies to enhance the innate protective
capacity of the lungs. The purpose of this symposium
was to present current research pertaining to the specific role of SPs and reactive oxygen-nitrogen species in
innate immunity. The symposium focused on the mechanisms of NO䡠-mediated toxicity for bacterial, human,
and animal models of SP-A- and NO䡠-mediated pathogen killing, microbial defense mechanisms against reactive oxygen-nitrogen species, specific examples and
signaling pathways involved in SP-A-mediated killing
of pulmonary pathogens, the structure and binding of
SP-A and SP-D to bacterial targets, and the immunoregulatory functions of SP-A.
L519
EB2001 SYMPOSIUM REPORT
ENZYMES THAT PREVENT AND REPAIR
PEROXYNITRITE-MEDIATED INJURY: STUDIES
WITH MICROBIAL PATHOGENS3
Knockout mice have helped to identify infections
against which iNOS or phox constitutes an essential,
nonredundant host defense (35). Generation of mice
deficient in both iNOS and phox reveals that these two
enzymes also constitute mutually redundant defenses,
to the extent that without them, the species could not
survive in the wild (47). Thus the antimicrobial effects
of iNOS are more widespread than revealed by the
iNOS-deficient mouse. Much of the antimicrobial action of iNOS may be mediated by formation of OONO⫺.
If we can identify enzymes that prevent or repair
OONO⫺-mediated damage, we can use the phenotype
of bacteria deficient in these enzymes as a probe to
3
gauge the importance of OONO⫺-mediated antibacterial effects.
Three levels of enzymatic defense against OONO⫺
have been identified. The first level is prevention. Superoxide dismutase and nitric oxide dioxygenase can
draw off the precursors of OONO⫺, preventing its formation. The second level is catabolism. Salmonella
typhimurium deficient in the peroxiredoxin alkyl hydroperoxide reductase subunit C (AhpC) are sensitive
to killing by NO2⫺ and S-nitrosoglutathione (GSNO) (6).
The peroxiredoxin from M. tuberculosis protects transfected human cells from autotoxicity caused by iNOS
(6). Yet AhpC does not detectably react with NO䡠 or
GSNO (5). Instead, Nathan’s laboratory has found that
peroxiredoxins from S. typhimurium, M. tuberculosis,
and Helicobacter pylori act as ONOO⫺ reductases,
breaking down OONO⫺ fast enough to protect bystander molecules from oxidation. The mechanism of
ONOO⫺ breakdown is the reversible oxidation of active-site Cys to sulfonic acid followed by intramolecular
disulfide bond formation and reduction either by a
dedicated flavoprotein (AhpF) or by thioredoxin (TRX).
TRX, in turn, is reduced by its own dedicated flavoprotein, TRX reductase. Tyr nitration is a much less sensitive indicator of the reaction of AhpC with OONO⫺
than is oxidation of the acidic active-site Cys (5). These
findings imply that NO2⫺, GSNO, and iNOS can cause
cytotoxicity via generation of OONO⫺. The third level
is repair. Nathan’s laboratory demonstrated a markedly increased sensitivity to NO2⫺ and GSNO in bacteria made deficient in a pathway that reverses a particular form of protein oxidation. However, neither NO2⫺
nor GSNO by itself causes this form of oxidation. Instead, they appear to be converted within the bacterial
cell into more potent oxidants. Evidence will be presented that this oxidant is likely to be OONO⫺. Thus
some reactive nitrogen intermediates (RNIs) may kill
bacteria by giving rise in the cell to OONO⫺, which
oxidizes critical amino acid residues. The enzyme that
reverses this oxidation may be the first example of an
enzyme that repairs OONO⫺-mediated injury.
Agents that inhibit the RNI resistance mechanisms
of pathogens may improve immunity in those diseases
for which RNIs represent an important but imperfect
element of host control. Mammalian homologs of these
enzymes may be important determinants of inflammatory tissue damage.
ROLE OF SP-A IN HOST DEFENSE
AGAINST MYCOBACTERIAL INFECTION4
Previous studies (15, 41, 54) have shown that SP-A
can bind specifically to mycobacteria, including M. tuberculosis, bacillus Calmette-Guerin (BCG), and Mycobacterium avium. The role that SP-A binding to BCG
plays in host defense against this organism has been
investigated with rat macrophages and BCG as a
model system. SP-A enhances entry of BCG into macrophages approximately fivefold. This enhanced entry
4
Presented by Carl Nathan.
AJP-Lung Cell Mol Physiol • VOL
Presented by Virginia L. Shepherd.
281 • SEPTEMBER 2001 •
www.ajplung.org
Downloaded from http://ajplung.physiology.org/ by 10.220.33.6 on June 18, 2017
tion by ⬃40% and stimulates a significant decrease in
mycoplasmal numbers (19). These data have been confirmed in vivo because transgenic mice deficient in
SP-A as well as mice lacking the inducible form of
nitric oxide synthase (iNOS) were shown to clear mycoplasmas less efficiently (17).
Although SP-A modulates NO䡠 production, this may
not be the primary mechanism by which SP-A mediates bacterial killing. In vitro assays of NO䡠-mediated
bacterial killing have been performed in the absence of
AMs, with chemical donors as a NO䡠 source. Data from
these experiments have demonstrated that NO䡠 itself is
rarely toxic to bacteria but that high concentrations of
NO䡠 congeners such as ONOO⫺ (formed at diffusion
rates by the interaction of superoxide with NO䡠) are
very efficient at bacterial killing (4, 17). Levels of NO䡠
produced by AMs (as measured from nitrate and nitrite
levels in culture medium) are much lower than those
required for bacterial killing by NO䡠 donors in the absence of AMs; however, calculations based on the cellular
rates of NO䡠 production and the size of the phagolysosome have indicated that the high concentrations of
ONOO⫺ necessary for bacterial killing (up to 500 ␮M)
are possible within this microenvironment (11).
SP-A has been shown to increase intracellular Ca2⫹
levels in rat AMs, an effect that is necessary for phagocytosis of pathogens (40). An increase in intracellular
Ca2⫹ is also required for the translocation of the
p47phox component of phox from the cytoplasm to the
membrane for the production of superoxide (14). Therefore, several possibilities exist to link SP-A together
with NO䡠 for efficient bacterial killing, which are not
mutually exclusive. 1) SP-A and bacterial lipopolysaccharide (LPS) act synergistically to upregulate NO䡠
production to levels toxic to bacteria. 2) SP-A stimulates an increase in intracellular Ca2⫹ levels, triggering phagocytosis of pathogens and direction to the
phagolysosomal compartment. 3) SP-A-mediated increases in intracellular Ca2⫹ levels induce assembly of
the phox complex, allowing for superoxide and ultimately ONOO⫺ production.
L520
EB2001 SYMPOSIUM REPORT
STRUCTURAL DETERMINANTS OF PULMONARY
COLLECTINS AFFECTING INTERACTIONS
WITH MICROBIAL LIGANDS5
SP-A and SP-D are Ca2⫹-binding lectins with a discrete four-domain primary structure consisting of a
5
Presented by Dennis R. Voelker.
AJP-Lung Cell Mol Physiol • VOL
disulfide-forming NH2 terminus, a collagen-like region,
a coiled neck region, and a COOH-terminal carbohydrate recognition domain (CRD) (24). Each of these
domains has been implicated in important functions of
the proteins. The NH2-terminal region controls the
extent of covalent oligomerization and greatly amplifies the valence of trimeric collectin subunits. The
collagen-like domain and the neck region participate in
noncovalent trimerization of the pulmonary collectins
that dictates the fundamental trimeric valence of the
CRDs. The CRD itself is a major binding site for the
recognition of multiple microorganisms including yeast,
bacteria, and viruses (9, 29). Most of the activity of the
CRD is attributable to the specific recognition of the
position 3 and 4 hydroxyls of mannose- and glucosecontaining carbohydrates (16). Mounting evidence has
indicated that these proteins are important primary
interacting elements of the innate immune system
in the alveolar space of the lung (9, 29, 56). Voelker’s
laboratory (24, 31, 32, 37–39, 43) has assembled a
large collection of point and domain mutants to probe
the structure and function of these proteins in interactions with microorganisms and their surface ligands.
Two classes of ligands currently under investigation
are rough LPS and mycoplasma membrane components. Both classes of ligand can be examined for
binding to the pulmonary collectins with solid-phase
assays or intact organisms.
Several important general principles have emerged
from these studies. One surprising finding is that the
ability of the collectins to bind to an affinity matrix
such as mannose-Sepharose is a poor predictive indicator of microbial recognition. A striking example
of this occurs with a tandem mutant of SP-D
(E321Q,N323D), which despite losing the capacity to
bind a mannose-Sepharose affinity matrix (38), exhibits enhanced binding to rough LPS ligands. Additional
findings demonstrate that the oligomerization attributable to the collagen domains of SP-A and SP-D is also
required for high-affinity LPS recognition but is not
essential for high-affinity recognition of carbohydrate
matrices or phospholipid ligands. The covalent oligomerization of SP-A has also been shown to be essential for
LPS recognition but not for carbohydrate recognition
per se (32). Several mutations within the CRD of SP-A
are also silent with regard to phospholipid and carbohydrate recognition (43) but can cause profound defects
in LPS recognition. Such findings clearly indicate that
modified forms of the collectins with either enhanced or
diminished capacity to recognize pathogens can be
generated without destroying other simple ligand recognition properties.
Direct comparison of LPS recognition and Mycoplasma pneumoniae ligand recognition also provides
unexpected results. Experiments with several structural mutants of the pulmonary collectins reveal that it
is possible to produce proteins with a selectively elevated or reduced affinity for one microbial class but
that have different binding characteristics for a second
microbial class. Thus SP-D variants with enhanced
LPS recognition can exhibit complete loss of myco-
281 • SEPTEMBER 2001 •
www.ajplung.org
Downloaded from http://ajplung.physiology.org/ by 10.220.33.6 on June 18, 2017
is accompanied by an SP-A-mediated induction of
BCG-induced NO䡠 and tumor necrosis factor (TNF)-␣
production (55). SP-A alone does not stimulate these
macrophages to produce either mediator in the absence
of BCG. SP-A opsonization of BCG also results in
decreased intracellular survival of BCG, which is also
dependent on the production of NO䡠 (55).
At least three different pathways or models could explain this ability of SP-A to increase both entry of BCG
into the macrophage and signaling for production of NO䡠:
1) SP-A might bind to a specific cell surface receptor and
increase expression of BCG receptors, 2) SP-A could in
some way present BCG to a BCG receptor in such a way
as to enhance uptake, and 3) SP-A-BCG complexes might
interact with a receptor that recognizes the complex and
mediates both signaling and internalization. In a previous report (7), a SP-A-specific receptor of 210-kDa molecular mass (SPR210) was isolated and characterized. It
was later shown that antibodies against this receptor
blocked SP-A-BCG-induced mediator production and increased BCG entry in the presence of SP-A (54). These
results support the following model: SP-A alone can bind
to SPR210, but this binding does not result in signaling.
However, when complexed to BCG, the SPR210 can now
mediate not only phagocytosis of the SP-A-BCG complex
but can also initiate signaling for NO䡠 production (55).
Recent studies from Shepherd’s laboratory have pursued the hypothesis that the binding of SP-A-BCG complexes to the SPR210 initiates specific intramacrophage
signaling pathways leading to enhanced BCG killing.
Treatment of macrophages with an inhibitor of tyrosine
kinases (herbimycin A) blocks NO䡠 production induced by
BCG and SP-A-BCG interaction with macrophages. Furthermore, blockade of tyrosine kinase activation blocks
intramacrophage killing of BCG internalized in the presence or absence of SP-A. Downstream targets of tyrosine
phosphorylation are members of the mitogen-activated
protein kinase family, extracellular signal-regulated kinases 1 and 2. SP-A opsonization of BCG specifically
enhances phosphorylation and activation of these kinases. Finally, synthesis of iNOS is dependent on the
activation of the transcription factor nuclear factor (NF)␬B. Opsonization of BCG leads to increased activation of
NF-␬B, suggesting that this factor plays a role in SP-Aenhanced BCG killing.
In summary, SP-A plays a key role in binding to and
enhancing the uptake of BCG by macrophages. This
increased uptake is accompanied by decreased survival of
the internalized mycobacteria, and this killing mechanism stimulated by SP-A is mediated through the activation of a signaling pathway involving protein tyrosine
kinases, mitogen-activated protein kinases, and NF-␬B.
L521
EB2001 SYMPOSIUM REPORT
plasma recognition. Conversely, additional SP-D mutants have been identified that fail to bind LPS but
retain significant mycoplasma binding. These structure and function studies with the collectins indicate
that variants of these proteins can be engineered to
display selective interactions with specific pathogens.
This information may be particularly useful for interfering with pathogens that use their interaction with
the wild-type collectins to evade detection by the immune system.
IMMUNOMODULATORY FUNCTIONS
OF LUNG SURFACTANT6
6
Presented by Jo Rae Wright.
AJP-Lung Cell Mol Physiol • VOL
REFERENCES
1. Anstey NM, Weinberg JB, Hassanali MY, Mwaikambo ED,
Manyenga D, Misukonis MA, Arnelle DR, Hollis D, McDonald MI, and Granger DL. Nitric oxide in Tanzanian children with malaria: inverse relationship between malaria severity and nitric oxide production/nitric oxide synthase type 2
expression. J Exp Med 184: 557–567, 1996.
2. Blau H, Riklis S, van Iwaarden JF, McCormack FX, and
Kalina M. Nitric oxide production by rat alveolar macrophages
can be modulated in vitro by surfactant protein A. Am J Physiol
Lung Cell Mol Physiol 272: L1198–L1204, 1997.
3. Borron P, McIntosh JC, Korfhagen TR, Whitsett JA, Taylor J, and Wright JR. Surfactant-associated protein A inhibits
LPS-induced cytokine and nitric oxide production in vivo. Am J
Physiol Lung Cell Mol Physiol 278: L840–L847, 2000.
4. Brunelli L, Crow JP, and Beckman JS. The comparative
toxicity of nitric oxide and peroxynitrite to Escherichia coli. Arch
Biochem Biophys 316: 327–334, 1995.
5. Bryk R, Griffin P, and Nathan C. Peroxynitrite reductase
activity of bacterial peroxiredoxins. Nature 407: 211–215, 2000.
6. Chen L, Xie QW, and Nathan C. Alkyl hydroperoxide reductase subunit C (AhpC) protects bacterial and human cells
against reactive nitrogen intermediates. Mol Cell 1: 795–805,
1998.
7. Chroneos ZC, Abdolrasulnia R, Whitsett JA, Rice WR, and
Shepherd VL. Purification of a cell-surface receptor for surfactant protein A. J Biol Chem 271: 16375–16383, 1996.
8. Crouch E, Hartshorn K, and Ofek I. Collectins and pulmonary innate immunity. Immunol Rev 173: 52–65, 2000.
9. Crouch EC. Collectins and pulmonary host defense. Am J
Respir Cell Mol Biol 19: 177–201, 1998.
281 • SEPTEMBER 2001 •
www.ajplung.org
Downloaded from http://ajplung.physiology.org/ by 10.220.33.6 on June 18, 2017
Both SP-A and SP-D have been shown to regulate a
variety of immune cell functions including phagocytosis, chemotaxis, and production of reactive species and
cytokines (reviewed in Refs. 8, 56). The effects of these
proteins on innate immune cell functions are, in some
cases, controversial. For example, SP-A has been
shown to both enhance and inhibit the production of
TNF-␣ and NO䡠 metabolites (reviewed in Refs. 8, 56).
The reasons for these disparate findings are not
known, but Wright’s laboratory speculates that they
could involve a number of factors including the state of
activation of the cell, the type of cell, and the type of
stimulus or pathogen presented to the cell. Recent
studies suggest that the state of activation of the cell
may affect the response to SP-A. For example, SP-A
enhances LPS-induced production of NO䡠 metabolites
by interferon (IFN)-␥-treated AMs (49). In contrast,
SP-A inhibits LPS-induced production of nitrate and
nitrite in AMs that are not treated with IFN-␥ (49).
Similar results are obtained when the levels of iNOS
enzyme are analyzed by Western blot; SP-A reduces
the levels of immunoreactive iNOS in cells treated with
LPS and enhances iNOS expression levels in cells
treated with IFN-␥.
It is also possible that different cells may have different responses to SP-A. Recent studies have demonstrated that the response of specific cells lines to SP-A
does not always duplicate the response of AMs (Wright,
unpublished data). Likewise, SP-A stimulates the chemotaxis of AMs but not of monocytes (51). Indirect
evidence from several laboratories suggests that SP-A
may exert cell-specific effects. For example, SP-A enhances production of TNF-␣ by THP-1 cells (22) but
inhibits production by AMs (33, 44) and U937 cells (46)
depending on the type of stimulus utilized. In vivo
studies have shown that SP-A-deficient mice have
higher levels of proinflammatory cytokines and nitrite
in their lavage fluid after intratracheal challenge with
bacteria (25, 27), viruses (26, 28), or LPS (3) compared
with wild-type mice. Thus these in vivo studies suggest
that SP-A serves a protective role in pulmonary host
defense.
Both indirect and direct evidence suggest that SP-A
and SP-D may elicit differential effects depending on
the type of pathogen or stimulus presented to the cells.
For example, Sano et al. (46) reported that SP-A has
differential effects on LPS-mediated production of
TNF-␣ by U937 cells depending on whether the cells
are stimulated with a rough or smooth serotype of LPS.
SP-A inhibited production of TNF-␣ induced by smooth
LPS, a finding consistent with a previous study (33)
and an in vivo study using SP-A-deficient mice (3). In
contrast, SP-A had no effect on production of TNF-␣
induced by a rough serotype of LPS (46). The mechanism of this differential effect involved the ability of
SP-A to differentially regulate the binding of LPS to
CD14 (45, 46). Indirect evidence from a variety of
different investigations is also consistent with the possibility that different pathogens elicit different effects.
For example, SP-A inhibits the production of NO䡠 metabolites in the presence of M. tuberculosis (42), and
this inhibition is correlated with the reduced killing of
organisms in the presence SP-A. In contrast, SP-A
enhances the production of NO䡠 by both monocytederived macrophages in the presence of BCG and
mouse AMs in the presence of M. pulmonis (17, 19).
Thus all of these studies considered in aggregate
suggest that the widely differing effects of SP-A that
have been reported may be at least partially explained
by the responding cell type, the pathogen that is utilized, or the state of activation of the cell. A complete
understanding of the modulation of the inflammatory
response by SP-A will require in vitro studies to elucidate the mechanisms of regulation at the cellular and
molecular levels and in vivo studies using mice with
targeted gene deletions in SPs, cytokines, and their
receptors to elucidate the overall contribution of these
proteins to the immune status in the lung.
L522
EB2001 SYMPOSIUM REPORT
AJP-Lung Cell Mol Physiol • VOL
31.
32.
33.
34.
35.
36.
37.
38.
39.
40.
41.
42.
43.
44.
45.
46.
47.
49.
in experimental salmonellosis. II. Effects on microbial proliferation and host survival in vivo. J Exp Med 192: 237–248, 2000.
McCormack FX, Kuroki Y, Stewart JJ, Mason RJ, and
Voelker DR. Surfactant protein A amino acids Glu195 and
Arg197 are essential for receptor binding, phospholipid aggregation, regulation of secretion, and the facilitated uptake of phospholipid by type II cells. J Biol Chem 269: 29801–29807, 1994.
McCormack FX, Pattanajitvilai S, Stewart J, Possmayer
F, Inchley K, and Voelker DR. The Cys6 intermolecular
disulfide bond and the collagen-like region of rat SP-A play
critical roles in interactions with alveolar type II cells and
surfactant lipids. J Biol Chem 272: 27971–27979, 1997.
McIntosh JC, Mervin-Blake S, Conner E, and Wright JR.
Surfactant protein A protects growing cells and reduces TNF-␣
activity from LPS-stimulated macrophages. Am J Physiol Lung
Cell Mol Physiol 271: L310–L319, 1996.
Nathan C. Inducible nitric oxide synthase: what difference does
it make? J Clin Invest 100: 2417–2423, 1997.
Nathan C and Shiloh MU. Reactive oxygen and nitrogen
intermediates in the relationship between mammalian hosts and
microbial pathogens. Proc Natl Acad Sci USA 97: 8841–8848,
2000.
Nozaki Y, Hasegawa Y, Ichiyama S, Nakashima I, and
Shimokata K. Mechanism of nitric oxide-dependent killing of
Mycobacterium bovis BCG in human alveolar macrophages. Infect Immun 65: 3644–3647, 1997.
Ogasawara Y, McCormack FX, Mason RJ, and Voelker DR.
Chimeras of surfactant proteins A and D identify the carbohydrate recognition domains as essential for phospholipid interaction. J Biol Chem 269: 29785–29792, 1994.
Ogasawara Y and Voelker DR. Altered carbohydrate recognition specificity engineered into surfactant protein D reveals
different binding mechanisms for phosphatidylinositol and glucosylceramide. J Biol Chem 270: 14725–14732, 1995.
Ogasawara Y and Voelker DR. The role of the amino-terminal
domain and the collagenous region in the structure and the
function of rat surfactant protein D. J Biol Chem 270: 19052–
19058, 1995.
Ohmer-Schrock D, Schlatterer C, Plattner H, and Schlepper-Schafer J. Lung surfactant protein A (SP-A) activates a
phosphoinositide/calcium signaling pathway in alveolar macrophages. J Cell Sci 108: 3695–3702, 1995.
Pasula R, Downing JF, Wright JR, Kachel DL, Davis TE,
and Martin WJ. Surfactant protein A (SP-A) mediates attachment of Mycobacterium tuberculosis to murine alveolar macrophages. Am J Respir Cell Mol Biol 17: 209–217, 1997.
Pasula R, Wright JR, Kachel DL, and Martin WJ. Surfactant protein A suppresses reactive nitrogen intermediates by
alveolar macrophages in response to Mycobacterium tuberculosis. J Clin Invest 103: 483–490, 1999.
Pattanajitvilai S, Kuroki Y, Tsunezawa W, McCormack
FX, and Voelker DR. Mutational analysis of Arg197 of rat
surfactant protein A. His197 creates specific lipid uptake defects.
J Biol Chem 273: 5702–5707, 1998.
Rosseau S, Hammerl P, Maus U, Gunther A, Seeger W,
Grimminger F, and Lohmeyer J. Surfactant protein A downregulates proinflammatory cytokine production evoked by Candida albicans in human alveolar macrophages and monocytes.
J Immunol 163: 4495–4502, 1999.
Sano H, Chiba H, Iwaki D, Sohma H, Voelker DR, and
Kuroki Y. Surfactant proteins A and D bind CD14 by different
mechanisms. J Biol Chem 275: 22442–22451, 2000.
Sano H, Sohma H, Muta T, Nomura S, Voelker DR, and
Kuroki Y. Pulmonary surfactant protein A modulates the cellular response to smooth and rough lipopolysaccharides by interaction with CD14. J Immunol 163: 387–395, 1999.
Shiloh MU, MacMicking JD, Nicholson S, Brause JE, Potter S, Marino M, Fang F, Dinauer M, and Nathan C. Phenotype of mice and macrophages deficient in both phagocyte
oxidase and inducible nitric oxide synthase. Immunity 10: 29–
38, 1999.
Stamme C, Walsh E, and Wright JR. Surfactant protein A
differentially regulates IFN-gamma- and LPS-induced nitrite
281 • SEPTEMBER 2001 •
www.ajplung.org
Downloaded from http://ajplung.physiology.org/ by 10.220.33.6 on June 18, 2017
10. De Groote MA, Granger D, Xu Y, Campbell G, Prince R,
and Fang FC. Genetic and redox determinants of nitric oxide
cytotoxicity in a Salmonella typhimurium model. Proc Natl Acad
Sci USA 92: 6399–6403, 1995.
11. Denicola A, Rubbo H, Rodriguez D, and Radi R. Peroxynitrite-mediated cytotoxicity to Trypanosoma cruzi. Arch Biochem
Biophys 304: 279–286, 1993.
12. Eifuku R, Yoshimatsu T, Yoshino I, Takenoyama M, Imahayashi S, So T, Hanagiri T, Nomoto K, and Yasumoto K.
Heterogeneous response patterns of alveolar macrophages from
patients with lung cancer by stimulation with interferongamma. Jpn J Clin Oncol 30: 295–300, 2000.
13. Fang FC. Perspectives series: host/pathogen interactions.
Mechanisms of nitric oxide-related antimicrobial activity. J Clin
Invest 99: 2818–2825, 1997.
14. Forman HJ, Zhou H, Gozal E, and Torres M. Modulation of
the alveolar macrophage superoxide production by protein phosphorylation. Environ Health Perspect 106, Suppl 5: 1185–1190,
1998.
15. Gaynor CD, McCormack FX, Voelker DR, McGowan SE,
and Schlesinger LS. Pulmonary surfactant protein A mediates
enhanced phagocytosis of Mycobacterium tuberculosis by a direct
interaction with human macrophages. J Immunol 155: 5343–
5351, 1995.
16. Hakansson K and Reid KB. Collectin structure: a review.
Protein Sci 9: 1607–1617, 2000.
17. Hickman-Davis J, Gibbs-Erwin J, Lindsey JR, and Matalon S. Surfactant protein A mediates mycoplasmacidal activity
of alveolar macrophages by production of peroxynitrite. Proc
Natl Acad Sci USA 96: 4953–4958, 1999.
19. Hickman-Davis JM, Lindsey JR, Zhu S, and Matalon S.
Surfactant protein A mediates mycoplasmacidal activity of alveolar macrophages. Am J Physiol Lung Cell Mol Physiol 274:
L270–L277, 1998.
20. Jesch NK, Dorger M, Enders G, Rieder G, Vogelmeier C,
Messmer K, and Krombach F. Expression of inducible nitric
oxide synthase and formation of nitric oxide by alveolar macrophages: an interspecies comparison. Environ Health Perspect
105, Suppl 5: 1297–1300, 1997.
21. Kalina M, Blau H, Riklis S, and Hoffman V. Modulation of
nitric oxide production by lung surfactant in alveolar macrophages. Adv Exp Med Biol 479: 37–48, 2000.
22. Kremlev SG, Umstead TM, and Phelps DS. Surfactant protein A regulates cytokine production in the monocytic cell line
THP-1. Am J Physiol Lung Cell Mol Physiol 272: L996–L1004,
1997.
23. Kroncke KD, Fehsel K, and Kolb-Bachofen V. Inducible
nitric oxide synthase in human diseases. Clin Exp Immunol 113:
147–156, 1998.
24. Kuroki Y and Voelker DR. Pulmonary surfactant proteins.
J Biol Chem 269: 25943–25946, 1994.
25. LeVine AM, Bruno MD, Huelsman KM, Ross GF, Whitsett
JA, and Korfhagen TR. Surfactant protein A-deficient mice
are susceptible to group B streptococcal infection. J Immunol
158: 4336–4340, 1997.
26. LeVine AM, Gwozdz J, Stark J, Bruno M, Whitsett J, and
Korfhagen T. Surfactant protein-A enhances respiratory syncytial virus clearance in vivo. J Clin Invest 103: 1015–1021,
1999.
27. LeVine AM, Kurak KE, Bruno MD, Stark JM, Whitsett JA,
and Korfhagen TR. Surfactant protein-A-deficient mice are
susceptible to Pseudomonas aeruginosa infection. Am J Respir
Cell Mol Biol 19: 700–708, 1998.
28. LeVine AM, Whitsett JA, Gwozdz JA, Richardson TR,
Fisher JH, Burhans MS, and Korfhagen TR. Distinct effects
of surfactant protein A or D deficiency during bacterial infection
on the lung. J Immunol 165: 3934–3940, 2000.
29. Mason RJ, Greene K, and Voelker DR. Surfactant protein A
and surfactant protein D in health and disease. Am J Physiol
Lung Cell Mol Physiol 275: L1–L13, 1998.
30. Mastroeni P, Vazquez-Torres A, Fang FC, Xu Y, Khan S,
Hormaeche CE, and Dougan G. Antimicrobial actions of the
NADPH phagocyte oxidase and inducible nitric oxide synthase
L523
EB2001 SYMPOSIUM REPORT
50.
51.
52.
53.
production by rat alveolar macrophages. Am J Respir Cell Mol
Biol 23: 772–779, 2000.
Steiner P, Efferen L, Durkin HG, Joseph GK, and Nowakowski M. Nitric oxide production by human alveolar macrophages in pulmonary disease. Ann NY Acad Sci 797: 246–249,
1996.
Tino MJ and Wright JR. Surfactant proteins A and D specifically stimulate directed actin-based responses in alveolar macrophages. Am J Physiol Lung Cell Mol Physiol 276: L164–L174,
1999.
Vazquez-Torres A and Fang FC. Oxygen-dependent antiSalmonella activity of macrophages. Trends Microbiol 9: 29–33,
2001.
Vazquez-Torres A, Jones-Carson J, Mastroeni P, Ischiropoulos H, and Fang FC. Antimicrobial actions of the NADPH
phagocyte oxidase and inducible nitric oxide synthase in experimental salmonellosis. I. Effects on microbial killing by activated
peritoneal macrophages in vitro. J Exp Med 192: 227–236, 2000.
54. Weikert LF, Edwards K, Chroneos ZC, Hager C, Hoffman L, and Shepherd VL. SP-A enhances uptake of bacillus
Calmette-Guerin by macrophages through a specific SP-A
receptor. Am J Physiol Lung Cell Mol Physiol 272: L989–
L995, 1997.
55. Weikert LF, Lopez JP, Abdolrasulnia R, Chroneos ZC, and
Shepherd VL. Surfactant protein A enhances mycobacterial
killing by rat macrophages through a nitric oxide-dependent
pathway. Am J Physiol Lung Cell Mol Physiol 279: L216–L223,
2000.
56. Wright JR. Immunomodulatory functions of surfactant. Physiol
Rev 77: 931–962, 1997.
Downloaded from http://ajplung.physiology.org/ by 10.220.33.6 on June 18, 2017
AJP-Lung Cell Mol Physiol • VOL
281 • SEPTEMBER 2001 •
www.ajplung.org