EB2001 SYMPOSIUM REPORT
Lung surfactant and reactive oxygen-nitrogen species: antimicrobial activity and host-pathogen interactions

Judy M. Hickman-Davis1, Ferric C. Fang2, Carl Nathan3, Virginia L. Shepherd4, Dennis R. Voelker5, and Jo Rae Wright6

1 Department of Anesthesiology, University of Alabama at Birmingham, Birmingham, Alabama 35249; 2 Clinical Microbiology Laboratory, University of Colorado Health Sciences Center, Denver 80262; 5 Department of Medicine, National Jewish Center for Immunology and Respiratory Medicine, Denver, Colorado 80206; 3 Department of Microbiology and Immunology, Weill Medical College of Cornell University, New York, New York 10021; 4 Department of Medicine, Vanderbilt University School of Medicine, Nashville, Tennessee 37212; and 6 Department of Cell Biology, Duke University, Durham, North Carolina 27710


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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·) is a well-recognized microbicidal product of macrophages, the mechanism(s) by which NO· 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·-mediated toxicity for bacterial, human, and animal models of SP-A- and NO·-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


    INTRODUCTION
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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),    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 macrophages (AMs). Superoxide, produced by the membrane-bound NADPH phagocyte oxidase (phox) of macrophages, combines with nitric oxide (NO·) to form the strong oxidant and bactericidal compound peroxynitrite (ONOO-) at near the diffusion limit for these two molecules.

Although NO· is a well-recognized microbicidal molecule of macrophages, the mechanism(s) by which NO· contributes to host defense remains undefined. NO· 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·-mediated toxicity for bacterial, human, and animal models of SP-A- and NO·-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.


    NITRIC OXIDE-MEDIATED MECHANISMS OF SALMONELLA KILLING BY MACROPHAGES1
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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· 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· donors have been shown to inhibit or kill diverse microbial species in vitro (13). Although the antimicrobial activity of NO· can be shown to be synergistic with that of reactive oxygen species, observations in Salmonella-infected murine peritoneal macrophages indicate that initial bacterial killing by phagocytes is primarily dependent on phox, with subsequent sustained inhibition of bacterial growth being mediated by NO· (47, 53). Similarly, in a murine systemic Salmonella infection model, phox appears to be essential for an initial reduction in organism burden, whereas NO· 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· targets in bacteria and relevant mechanisms of bacterial resistance to NO· 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
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The importance of NO· 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· production is differentially regulated in different species (20). The involvement of NO· 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·) 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· by human monocytes have failed, and the study of NO· 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· production by AMs has been demonstrated with transformed cell lines (21) and primary rat AMs (2). The importance of SP-A and NO· 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· but do not kill mycoplasmas; however, the addition of SP-A to these cells increases NO· production 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· production, this may not be the primary mechanism by which SP-A mediates bacterial killing. In vitro assays of NO·-mediated bacterial killing have been performed in the absence of AMs, with chemical donors as a NO· source. Data from these experiments have demonstrated that NO· itself is rarely toxic to bacteria but that high concentrations of NO· congeners such as ONOO- (formed at diffusion rates by the interaction of superoxide with NO·) are very efficient at bacterial killing (4, 17). Levels of NO· produced by AMs (as measured from nitrate and nitrite levels in culture medium) are much lower than those required for bacterial killing by NO· donors in the absence of AMs; however, calculations based on the cellular rates of NO· production and the size of the phagolysosome have indicated that the high concentrations of ONOO- necessary for bacterial killing (up to 500 µM) 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· for efficient bacterial killing, which are not mutually exclusive. 1) SP-A and bacterial lipopolysaccharide (LPS) act synergistically to upregulate NO· 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.


    ENZYMES THAT PREVENT AND REPAIR PEROXYNITRITE-MEDIATED INJURY: STUDIES WITH MICROBIAL PATHOGENS3
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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 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 NO<UP><SUB>2</SUB><SUP>−</SUP></UP> 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· 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 NO<UP><SUB>2</SUB><SUP>−</SUP></UP>, GSNO, and iNOS can cause cytotoxicity via generation of OONO-. The third level is repair. Nathan's laboratory demonstrated a markedly increased sensitivity to NO<UP><SUB>2</SUB><SUP>−</SUP></UP> and GSNO in bacteria made deficient in a pathway that reverses a particular form of protein oxidation. However, neither NO<UP><SUB>2</SUB><SUP>−</SUP></UP> 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
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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 is accompanied by an SP-A-mediated induction of BCG-induced NO· and tumor necrosis factor (TNF)-alpha 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· (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·: 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· 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· 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)-kappa B. Opsonization of BCG leads to increased activation of NF-kappa B, suggesting that this factor plays a role in SP-A-enhanced 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-kappa B.


    STRUCTURAL DETERMINANTS OF PULMONARY COLLECTINS AFFECTING INTERACTIONS WITH MICROBIAL LIGANDS5
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SP-A and SP-D are Ca2+-binding lectins with a discrete four-domain primary structure consisting of a 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 glucose-containing 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 mycoplasma 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
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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-alpha and NO· 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· metabolites by interferon (IFN)-gamma -treated AMs (49). In contrast, SP-A inhibits LPS-induced production of nitrate and nitrite in AMs that are not treated with IFN-gamma (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-gamma .

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-alpha 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-alpha by U937 cells depending on whether the cells are stimulated with a rough or smooth serotype of LPS. SP-A inhibited production of TNF-alpha 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-alpha 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· 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· by both monocyte-derived 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.


    FOOTNOTES

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: Judy.Hickman-Davis{at}ccc.uab.edu).

1  Presented by Ferric Fang.

2  Presented by Judy M. Hickman-Davis.

3  Presented by Carl Nathan.

4  Presented by Virginia L. Shepherd.

5  Presented by Dennis R. Voelker.

6  Presented by Jo Rae Wright.


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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[Abstract/Free Full Text].

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[Abstract/Free Full Text].

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[ISI][Medline].

5.   Bryk, R, Griffin P, and Nathan C. Peroxynitrite reductase activity of bacterial peroxiredoxins. Nature 407: 211-215, 2000[ISI][Medline].

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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[Abstract/Free Full Text].

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[Abstract/Free Full Text].

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, Suppl5: 1297-1300, 1997[ISI][Medline].

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[ISI][Medline].

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[Abstract/Free Full Text].

23.   Kroncke, KD, Fehsel K, and Kolb-Bachofen V. Inducible nitric oxide synthase in human diseases. Clin Exp Immunol 113: 147-156, 1998[ISI][Medline].

24.   Kuroki, Y, and Voelker DR. Pulmonary surfactant proteins. J Biol Chem 269: 25943-25946, 1994[Free Full Text].

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[Abstract].

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[Abstract/Free Full Text].

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[Abstract/Free Full Text].

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