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Surfactant Protein A Decreases Nitric Oxide Production by Macrophages in a Tumor Necrosis Factor-–Dependent Mechanism

Department of Medicine, Division of Pulmonary, Allergy, Critical Care and Occupational Medicine, Indiana University Medical Center, Indianapolis, Indiana; and Department of Cell Biology, Duke University Medical Center, Durham, North Carolina

Address correspondence to: William J. Martin II, M.D., College of Medicine, University of Cincinnati, Cincinnati, OH 45267. E-mail: william.martin{at}uc.edu

	Abstract

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Surfactant protein A (SP-A) modulates the lung defense system through regulation of cytokines and nitric oxide (NO) production by alveolar macrophages (AMs). Whether SP-A upregulates or downregulates production of proinflammatory cytokines and NO is controversial. This study demonstrates the molecular mechanism(s) by which SP-A suppresses NO production by activated murine AMs. NO production by interferon- (IFN-) and IFN- plus Mycobacterium avium–stimulated AMs was mediated through tumor necrosis factor- (TNF-) production, as addition of neutralizing anti–TNF- antibodies during AMs stimulation resulted in reduced NO production. SP-A suppressed NO production by activated AMs by inhibiting TNF- production. The maximum inhibitory effect of SP-A on NO production was observed at 20 µg/ml of SP-A concentration. Furthermore, SP-A inhibited activation of nuclear factor-B, a transcription factor required for induction of TNF- and inducible NO synthase genes. These findings suggest that SP-A suppresses NO production by activated AMs by inhibiting TNF- secretion and nuclear factor-B activation. This study also highlights the importance of SP-A levels in the lung, as changes in SP-A levels may modulate the local lung defense system.

Abbreviations: alveolar macrophages, AMs • bronchoalveolar lavage fluid, BALF • enzyme-linked immunosorbent assay, ELISA • electrophoretic mobility shift assay, EMSA • interferon-, IFN- • interleukin, IL • inducible nitric oxide synthase, iNOS • interferon regulatory factor-1, IRF-1 • lipopolysaccharide, LPS • nuclear factor-B, NF-B • nitric oxide, NO • peripheral blood mononuclear cells, PBMC • surfactant protein A, SP-A • tumor necrosis factor-, TNF-

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Surfactant protein A (SP-A) is the most abundant hydrophilic protein present in pulmonary surfactant and plays an important role in innate host defense (1). SP-A is primarily produced by type II alveolar epithelial cells, and belongs to a group of molecules termed collectins. Collectins are C-type lectins consisting of N-terminal collagenous and C-terminal carbohydrate binding domains. SP-A acts as a nonimmune opsonin, and enhances attachment and phagocytosis of a variety of pathogens, including Mycobacterium tuberculosis (M. tuberculosis), to alveolar macrophages (AMs) (2). Besides opsonization of microorganisms, SP-A also behaves like a cytokine and regulates transcription of genes encoding surfactant proteins (SP-A, SP-B, and SP-C), transcription factors (c-fos and c-Jun), and surfactant protein specific receptor in type II alveolar epithelial cells (3).

SP-A level increases in the lung of human immunodeficiency virus (HIV)-infected individuals (2). HIV-infected individuals are also susceptible to infection with opportunistic pathogens such as Mycobacterium avium (M. avium) (4). In the lung, AMs engulf M. avium and provide a safe haven for its survival and multiplication in the intracellular environment. M. avium survives in the AMs by controlling the local and systemic immune response. For example, M. avium inhibits interferon (IFN)-–induced macrophage activation by downregulating IFN- receptor, and the infected macrophages produce deactivating cytokines that further diminish the host response (5, 6). In vitro studies suggest that in the lung, SP-A interacts with AMs and modulates the lung defense system through production of cytokines, chemokines, and NO (7–13).

IFN- is primarily responsible for macrophage activation. IFN-–activated murine macrophages control mycobacterial growth through nitric oxide (NO) production (10, 13–15). NO production in macrophages is controlled by inducible nitric oxide synthase (iNOS), whose transcription in macrophages is regulated mainly by nuclear factor-B (NF-B) and interferon regulatory factor-1 (IRF-1) transcription factors (16, 17). M. avium stimulation enhances NF-B activation by producing proinflammatory cytokines such as tumor necrosis factor (TNF)- (18), whereas IRF-1 induction and activation is controlled by IFN- (16).

A role for SP-A in regulation of NO production by activated macrophages is controversial (8, 10, 12, 13). Many in vitro studies suggest a suppressive role for SP-A in production of proinflammatory cytokines and NO in response to lipopolysaccharide (LPS), M. tuberculosis, and Candida albicans (C. albicans)–stimulated AMs (7, 9–12). Recent in vivo findings also support the suppressive effect of SP-A by showing increased production of proinflammatory cytokines and NO in the lung of bacteria- and virus-infected or LPS-treated SP-A^-/- mice (19–23). In contrast, some in vitro work describes a stimulatory effect of SP-A in the production of proinflammatory cytokines and NO by activated monocytes and macrophages (8, 24, 25). This study was designed to explore the molecular mechanism(s) involved in SP-A–mediated modulation of NO production by murine AMs in response to IFN- and IFN- plus M. avium stimulation. Our results indicate that SP-A suppresses NO production by IFN-– and IFN- plus M. avium–stimulated murine AMs by inhibiting TNF- secretion and NF-B activation.

	Materials and Methods

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Animals
Pathogen-free female Swiss Webster mice (8–10 wk old) were purchased from Harlan Sprague Dawley (Indianapolis, IN). The mice were housed in groups of five in micro-isolation cages. Food and water were provided ad libitum.

Mycobacteria
M. avium (ATCC 25291) was grown in Middlebrook 7H9 broth (Difco Laboratories, Detroit, MI) at 37°C in 5% CO₂ until mid-log phase. Bacterial concentration was determined by Spectronic 20D (Milton Roy Co., Rochester, NY) and frozen in 1-ml aliquots in 10% glycerol until further use. Frozen aliquots were thawed and briefly sonicated before each use. Purity of the organism was determined before each use by staining with Kinyoun stain (Midatlantic Biomedical Inc., Paulsboro, NJ).

Media and Reagents
Dulbecco's modified Eagle's medium without phenol red was purchased from BioWhittaker (Walkersville, MD). Penicillin and streptomycin were obtained from Biofluids (Biosource International, Rockville, MD). Amphotericin B, polymyxin B, polymyxin B-agarose, glutamine, dithiothreitol (DTT), sodium orthovanadate (Na₃VO₄), sodium fluoride (NaF), aprotinin, ethylenediamine tetraacetic acid (EDTA), and phenylmethylsulfonyl fluoride (PMSF) were purchased from Sigma (St. Louis, MO). Nonidet P-40 (NP-40), Klenow enzyme, poly (dI-dC), and deoxyribonucleotides (dATP, dTTP, dGTP and dCTP) were obtained from Boehringer Mannheim (Indianapolis, IN). Recombinant murine IFN- and NF-B primers were purchased from Gibco BRL (Grand Island, NY). Murine TNF- enzyme linked immunosorbent assay (ELISA) kit, mouse TNF-–neutralizing antibodies, and normal goat immunoglobulin G (Ig G) were purchased from R&D systems (Minneapolis, MN). Affinity-purified polyclonal goat antibodies against NF-B p50 and NF-B p65 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA).

SP-A Isolation
Human SP-A was isolated from bronchoalveolar lavage fluid (BALF) of patients with alveolar proteinosis as previously described (26). Briefly, a surfactant pellet was obtained from BALF by centrifuging at 100,000 x g for 1 h. The pellet was extracted with butanol. The butanol-insoluble fraction was then suspended in buffer containing 30 mM octylglucoside, 150 mM NaCl, and 5 mM Tris, pH 7.4. Polymyxin B-agarose was added at 1:5 (vol/vol) to the suspension and incubated for 30 min at room temperature to remove contaminating endotoxin. The mixture was then dialyzed extensively against 5 mM Tris, pH 7.4, and SP-A was aspirated following centrifuging at 100,000 x g for 30 min. Purified SP-A used in this study contained very low levels of endotoxin (< 0.05 pg/µg of SP-A). The SP-A preparation used in this study failed to induce any TNF- secretion.

AM Isolation and Culture
AMs were isolated as previously described (10). Briefly, mice were killed by intraperitoneal injection of beuthanasia-D solution (Schering Plough Animal Health Corp., Kenilworth, NJ). A 20-gauge angiocatheter was inserted into the trachea proximal to the bifurcation. The lungs were lavaged 15 times with 0.8-ml aliquots of normal saline supplemented with 0.6 mM EDTA, penicillin (100 U/ml), streptomycin (100 µg/ml), and amphotericin B (0.5 µg/ml). The BALF was centrifuged (600 x g) for 10 min. The cell pellet was suspended in red blood cell lysing buffer (10 mM KHCO₃ and 150 mM NH₄Cl) and centrifuged again to pellet AMs. Cells were then suspended in Dulbecco's modified Eagle's medium supplemented with glutamine (300 µg/ml) and cultured at a density of 2.5 x 10⁵ cells per well in 96-well tissue culture plates for estimating NO production and cytokine secretion. For electrophoretic mobility shift assay (EMSA), cells were cultured at a density of 5 x 10⁶ cells per well in 6-well tissue culture plates. Cell viability and purity were greater than 97% as demonstrated by trypan blue exclusion and Diff-quick (Fisher Scientific, Chicago, IL) staining, respectively. All incubations were done at 37°C in 5% CO_2. Swiss Webster AMs were used in all experiments.

AM Stimulation
To examine the effect of IFN- priming in TNF- and NO production by AMs, cells were treated with IFN- (0–200 U/ml) for 48 h. However, the medium was replaced with fresh IFN- after 24 h. After the last treatment, culture supernatants were collected for NO and TNF- quantification. The triggering effect of M. avium on NO and TNF- production was examined by stimulating overnight IFN-–primed AMs with M. avium (10:1 to 100:1, M. avium to macrophage ratio) for 24 h. Effect of SP-A and IFN- on NO and TNF- production was examined by treating cells overnight with SP-A and IFN- and then triggering with M. avium. TNF- neutralization experiments were performed by adding anti–TNF- antibodies during M. avium stimulation of IFN-–primed AMs. In mobility shift experiments, macrophages were treated overnight with SP-A and then stimulated with M. avium and IFN- for 2–6 h.

Cytokines and NO Quantification
Secretion of TNF- in the cell-free culture supernatant was determined by murine TNF- ELISA kit following the manufacturer's instructions. IFN-–induced NO production is equally well estimated by measuring either nitrite or nitrate contents of the cell-free supernatant (10, 12). NO production in the cell-free supernatant of cultured AMs was estimated by measuring the amount of nitrite by the Griess assay as described previously (18). Briefly, 50 µl of macrophage culture supernatant was mixed with equal amount of Griess reagent (1% sulfanilamide, 0.1% naphthylenediamine hydrochloride, 2.5% H₃PO₄) in a 96-well tissue culture plate, and incubated for 10 min at room temperature. The absorbance at 540 nm was recorded with a Titertek microplate ELISA reader (Flow Laboratories, Mclean, VA). Nitrite concentrations were determined by the standard curve generated by incubating serial dilutions of sodium nitrite (0–250 µM) with Griess reagent.

Nuclear Extraction and EMSA
Nuclear extracts were prepared as described previously (5). Macrophages were washed twice with ice-cold phosphate-buffered saline and incubated on ice for 15 min in 400 µl hypotonic buffer containing 10 mM HEPES (pH 7.9), 10 mM KCl, 0.10 mM EDTA, 1 mM DTT, 0.5 mM PMSF, 1 µg/ml aprotinin, and 1 mM Na₃VO₄. The cells were then lysed by adding 25 µl of 10% NP-40 and brief vortexing. Nuclei were pelleted and extracted on ice for 15 min in 100 µl buffer containing 20 mM HEPES (pH 7.9), 25% glycerol, 0.4 M NaCl, 1 mM EDTA, 1 mM DTT, 0.5 mM PMSF, 1 µg/ml aprotinin, and 1 mM Na₃VO₄. Nuclear extracts were recovered from supernatants after centrifugation at 10,000 x g for 15 min. Protein concentration was determined by Bradford method using Bio-Rad protein assay reagent (Bio-Rad, Richmond, CA). The extracts were stored at –80°C until further use.

EMSA were performed in 20-µl binding reactions containing 3 µg of nuclear extract, 10 mM Tris HCl (pH 7.5), 50 mM NaCl, 1 mM DTT, 5 mM MgCl₂, 10% glycerol, 0.2% NP-40, 1 µg poly (dI-dC), and 70,000 cpm ³²P-dCTP labeled (Amersham, Arlington Heights, IL) NF-B probe radiolabeled by filling with Klenow DNA polymerase. The NF-B probe was derived from the murine iNOS promoter (27). The sequence of NF-B probe is 5'-GAAGCTTGGGGACTCTCCCTTTG-3'. Binding reactions were incubated for 20 min at room temperature and then subjected to electrophoresis on 6% nondenaturing polyacrylamide gels in 0.5x TBE (22.3 mM Tris-borate and 0.5 mM EDTA) buffer. The gels were dried and analyzed by autoradiography. In competition assays, 100x unlabeled NF-B probe was added along with radiolabeled NF-B probe. In super-shift assays, 2 µg of NF-B p50 and NF-B p65 antibodies were incubated with binding reactions 20 min before the addition of radiolabeled NF-B probe.

Statistical Analysis
The experiments were performed in triplicate. Results are expressed as means ± SEM. Statistical analysis of data was performed by t test using SigmaStat software at P < 0.05. Densitometric analysis of EMSA gels was performed using Bio-Rad densitometer and Bio-Rad multi-analyst software.

	Results

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IFN- Induces NO Production in Murine AMs in a TNF-–Dependent Mechanism
We examined NO production in murine AMs by stimulating with increasing concentrations of IFN- (0–200 U/ml) for 48 h. NO production was estimated by measuring the nitrite contents in the cell-free culture supernatant using Griess reagent. IFN-–induced NO production in a dose-dependent manner (Figure 1A). Maximum NO production occurred at 100 U/ml of IFN-. TNF- concentration in the culture supernatant of untreated AMs (94.5 ± 16.5 pg/ml) was also increased significantly in IFN- (100 U/ml)–stimulated AMs (1,308.3 ± 316 pg/ml; P < 0.05). To evaluate the role of TNF- in the production of NO in IFN-–stimulated AMs, neutralizing anti–TNF- antibodies (50 µg/ml) were added to the culture medium during IFN- stimulation. Addition of anti–TNF- antibodies reduced NO production in IFN-–stimulated AMs (10 ± 1.5 nmoles/2.5 x 10⁵ AMs to 5.9 ± 0.7 nmoles/2.5 x10⁵ AMs, P < 0.05). However, addition of isotype specific IgG during IFN- stimulation did not change NO production (Figure 1B).

View larger version (20K): [in this window] [in a new window]   Figure 1. IFN- induces NO production in murine AMs in a TNF-–dependent mechanism. (A) AMs (2.5 x 105) were stimulated with increasing concentrations of IFN- (0–200 U/ml) for 48 h. NO production was estimated by measuring nitrite contents in the cell-free culture supernatant using Griess reagent. IFN-–induced NO production in a dose-dependent manner. Maximum NO production occurred at 100 U/ml of IFN-. (B) AMs (2.5 x 105) were stimulated with IFN- (100 U/ml) for 48 h in presence or absence of neutralizing anti–TNF- antibodies (50 µg/ml) or isotype IgG (50 µg/ml). Addition of anti–TNF- antibodies reduced NO production significantly (P < 0.05) in IFN-–stimulated AMs. Addition of isotype IgG did not change NO production. NT, no treatment.

Priming of AMs with Low Concentration of IFN- Is Sufficient to Induce NO Production in M. avium–Stimulated Murine AMs

View larger version (25K): [in this window] [in a new window]   Figure 2. M. avium stimulation enhances NO production in IFN--primed AMs in a TNF-–dependent mechanism. (A) AMs (2.5 x 105) with or without IFN- priming (10 U/ml) for 18 h were incubated with increasing M. avium to macrophage ratio for 24 h at 37°C. Unprimed AMs did not induce NO production even at 100:1 M. avium to macrophage ratio of stimulation. NO production was increased with increasing M. avium to macrophage ratio of stimulation. Maximum NO production was observed at 50:1 ratio of stimulation. (B) AMs (2.5 x 105) primed with IFN- for 18 h were incubated with 20:1 M. avium to macrophage ratio of stimulation for 24 h in presence or absence of neutralizing anti–TNF- antibodies (50 µg/ml) and in the presence of anti–TNF- antibodies plus SP-A (20 µg/ml) or isotype IgG (50 µg/ml). Addition of anti–TNF- antibodies with or without SP-A reduced NO production significantly in IFN- plus M. avium–stimulated cells (P < 0.05). There was no additional effect of SP-A to the use of anti-TNF antibodies alone. Addition of isotype IgG did not change NO production. NT, no treatment; MA, M. avium.

SP-A Suppresses NO Production in IFN-– and IFN- plus M. avium–Stimulated AMs in a Concentration-Dependent Manner

View larger version (20K): [in this window] [in a new window]   Figure 3. SP-A inhibits NO production by IFN-– and IFN- plus M. avium–stimulated AMs in a concentration-dependent manner. AMs (2.5 x 105) were stimulated with IFN- (100 U/ml) and IFN- (10 U/ml) plus M. avium (20:1, M. avium to macrophage ratio) in presence or absence of SP-A (0–100 µg/ml) at 37°C for 48 h. NO production was determined. Maximum inhibition of NO production occurred at 20 µg/ml of SP-A in both IFN-– and IFN- plus M. avium–stimulated AMs. Percent inhibition was calculated by considering 0% inhibition of NO production by IFN-– and IFN- plus M. avium–stimulated AMs in the absence of SP-A. The results are mean ± SEM of three independent experiments performed in triplicate. MA, M. avium.

SP-A Suppresses NO Production in IFN-– and IFN- plus M. avium–Stimulated AMs by Inhibiting TNF- Secretion

View larger version (34K): [in this window] [in a new window]   Figure 4. Evidence for SP-A to inhibit NO production and TNF- secretion in IFN-– and IFN- plus M. avium–stimulated AMs. (A, B) AMs (2.5 x 105) were stimulated with IFN- (100 U/ml) in the presence or absence of SP-A (20 µg/ml) at 37°C for 48 h. NO and TNF- concentrations were determined. SP-A significantly reduced NO and TNF- secretion (P < 0.05, both comparisons). (C, D) IFN- (10 U/ml)– and SP-A (20 µg/ml)–treated AMs (2.5 x 105) were stimulated with M. avium in the presence or absence of SP-A for 24 h. NO and TNF- concentrations were determined. SP-A significantly reduced both NO and TNF- production (P < 0.05, both comparisons). NT, no treatment; MA, M. avium.

SP-A Inhibits NF-B Activation
M. avium stimulation results in NF-B activation through production of proinflammatory cytokines such as TNF-, which activates NF-B and enhances iNOS gene transcription. We examined the effect of SP-A on activation of NF-B in IFN--, M. avium–, and IFN- plus M. avium–stimulated AMs. Treatment of murine AMs with SP-A for 18 h before IFN- and M. avium stimulation resulted in reduced NF-B activation (Figures 5A, 5B, 6A, and 6B). Addition of anti–TNF- antibodies during IFN- stimulation resulted in inhibition of NF-B activation suggesting a requirement of TNF- in NF-B activation (Figure 5C). The inhibitory effect of SP-A on NF-B activation was moderate, but is well correlated with its inhibitory effect on NO production (Figures 4A and 4C) and TNF- secretion (Figures 4B and 4D).

View larger version (37K): [in this window] [in a new window]   Figure 5. SP-A inhibits NF-B activation in IFN-–stimulated AMs in a TNF-–dependent mechanism. (A) AMs (5 x 106) incubated overnight with SP-A (20 µg/ml) were treated with IFN- for 6 h. Nuclear extracts were prepared and incubated with 32P-dCTP–labeled NF-B sequence and binding assayed by EMSA gel. NF-B percent inhibition was calculated following densitometry. Optical density (OD) from respective non–SP-A–treated lane was used as a control (0% inhibition). (B) For competition experiments, nuclear extracts were incubated with 100-fold excess of unlabeled oligonucleotides. Nuclear extracts were pre-incubated with anti–NF-B (anti-p50 and anti-p65) antibodies for 20 min before addition of radiolabeled probe to identify the NF-B proteins. (C) AMs (5 x 106) were incubated with IFN- for 18 h in the presence or absence of anti–TNF- antibodies. Addition of anti–TNF- antibodies inhibited NF-B activation in IFN-–stimulated cells. These experiments are representative of three experiments.

View larger version (52K): [in this window] [in a new window]   Figure 6. SP-A inhibits NF-B activation in M. avium– and IFN- plus M. avium–stimulated AMs. (A) AMs (5 x 106) incubated with SP-A (20 µg/ml) for 18 h were stimulated with M. avium and M. avium plus IFN- for 2 h. NF-B percent inhibition was calculated following densitometry. OD from respective non–SP-A–treated lane was used as a control (0% inhibition). (B) For competition experiments, nuclear extracts were incubated with 100-fold excess of unlabeled oligonucleotides. Nuclear extracts were pre-incubated with anti–NF-B (anti-p50 and anti-p65) antibodies for 20 min before addition of radiolabeled probe to identify the NF-B proteins. These experiments are representative of three experiments.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

In vitro studies suggest a role for SP-A in modulating the lung defense system by regulating the release of inflammatory mediators by AMs (8–13, 23–25). The results of the present study indicate that SP-A reduces NO production by inhibiting TNF- release by IFN-– and IFN- plus M. avium–stimulated AMs. Decreased TNF- production by SP-A–treated rat AMs in response to LPS stimulation was initially reported by McIntosh and coworkers (7). Later, Sano and coworkers (9) observed a similar effect of SP-A on TNF- production in LPS-stimulated rat AMs and human U937 monocytic cells. Rosseau and colleagues (11) also reported a downregulatory effect of SP-A on the production of TNF- and other proinflammatory cytokines and chemokines in C. albicans–stimulated human AMs and peripheral blood mononuclear cells (PBMC). Also, that study describes an inhibitory effect of SP-A on TNF- gene transcription, which further support our data that describes inhibitory effect of SP-A on NF-B activation.

The suppressive effect of SP-A has been more precisely defined following the availability of SP-A^-/- mice. To our knowledge, the results of all in vivo studies favor the suppressive effect of SP-A on the production of proinflammatory cytokines and NO (20–23). Borron and coworkers (23) found an increase in TNF- and nitrite concentration in the BALF of intratracheally LPS-instilled SP-A^-/- mice. Others have also described an increase in NO, proinflammatory cytokines (TNF-, interleukin [IL]-1ß, IL-6), and chemokine production in lungs of bacterial- and viral-infected SP-A^-/- mice (20–22). SP-A–treated human PBMC also produce low amounts of IL-2 and TNF- following polyclonal activation (29–31). How SP-A inhibits production of those cytokines by PBMC is currently not known, but a similar effect of SP-A was also observed on the production of IL-8 by phorbol 12-myristate 13-acetate–stimulated human eosinophils (32). Taken together, all these studies reflect a global anti-inflammatory and immunosuppressive effect of SP-A on cells of the immune system, and tend to favor the results of the current investigation.

In contrast to our results, a stimulatory effect of SP-A on the production of proinflammatory cytokines has also been reported (24, 25). SP-A enhanced TNF- production in vitamin D₃–differentiated THP-1 cells, which peaked at 4 h and then declined. This suggests that SP-A have a stimulatory effect on TNF- production by vitamin D₃–differentiated THP-1 cells during first few hours of treatment. A previous report also describes a priming effect of vitamin D₃ in LPS-induced TNF- release by human monocytes (33). Failure of TNF- production under similar conditions by undifferentiated THP-1 cells further favors this notion (24). However, in both studies SP-A was purified by different methods compared with our study, and different SP-A preparations have different biological effects; this may also account for differences in the results (34).

The results of the current investigation indicate that SP-A inhibits activation of NF-B, which regulates TNF- and NO production in macrophages (17, 18). Reduced NF-B activation observed in our study may be responsible for the decreased TNF- and NO production in SP-A–treated AMs. This is consistent with a recent report by Murakami and colleagues (35) that shows reduced NF-B activity in human embryonic kidney 293 cells transiently transfected with the cDNA of Toll-like receptor 2 and stimulated with Staphylococcus aureus peptidoglycan in presence of SP-A. Interestingly, the inhibitory effect of SP-A on NF-B activation in human embryonic kidney 293 cells was moderate (30–40%), and is consistent with our results (Figures 5A and 6A). In contrast, Koptides and coworkers (36) observed an increase in NF-B activation in SP-A–treated THP-1 cells. It is important to note that those THP-1 cells were differentiated with vitamin D₃ for 72 h before SP-A treatment, and vitamin D₃ has been shown to modulate the sensitivity of THP-1 cells to LPS (33). Also, if SP-A–treated THP-1 cells were washed and then retreated with SP-A, NF-B activation was decreased (25). These results tend to support our findings, which demonstrate decreased NF-B activation and TNF- production in response to IFN- and M. avium stimulation in SP-A–treated cells.

The results of the present investigation show reduced NO production by SP-A–treated murine AMs in response either to IFN-– or IFN- plus M. avium–stimulation. Stamme and coworkers (12) also reported an inhibitory effect of SP-A on NO production by LPS-stimulated rat AMs. However, in the same study, they observed a stimulatory effect of SP-A on IFN-–stimulated rat AMs. This may be due to differential regulation of NO production by murine and rat AMs. Murine AMs require IFN- and LPS or microbial stimulation to induce NO production (Figure 2A), whereas only LPS stimulation or microbial infection is sufficient to induce NO production by rat macrophages (12, 13). This suggests that rat and murine AMs may differ in the induction and activation of transcription factors such as NF-B and IRF-1, which are required for NO production (16, 17). In murine macrophages, NF-B and IRF-1 are activated by TNF- and IFN-, respectively (16, 18). The inhibitory effect of SP-A on NO production by LPS-stimulated rat AMs may be due to decreased TNF- secretion (7, 23). However, the stimulatory effect of SP-A in IFN-–stimulated rat AMs may be due to increased sensitivity of rat AMs to IFN- stimulation, which may induce increased activation of IRF-1 and other transcription factors that bind to IFN- response element, -activated site, and IFN-stimulated response element in the iNOS promoter (16).

The inhibitory effect of SP-A on NO production by AMs may be detrimental in certain pulmonary infections, such as mycobacterial infections. NO is at least one of the mechanisms proposed to control mycobacterial growth (10, 14, 15). In the lung, SP-A is produced by type II alveolar epithelial cells and is an abundant protein in the alveolar lining fluid. The estimated concentration in the epithelial lining fluid of the alveoli is from 180 µg/ml to 1.8 mg/ml (1, 35), a range consistent with the concentrations of SP-A used in this study. During HIV infection, SP-A concentration increases in the lung several fold (2). HIV-infected individuals are susceptible to infection with opportunistic pathogens such as M. avium (4). In vitro data also indicate that SP-A enhances growth of M. tuberculosis in murine AMs (10). Altogether, this suggests that increased SP-A levels in the lungs of HIV-infected individuals may enhance mycobacterial pathogenesis as well as reduce the capability of AMs to produce NO, which is one of the mechanisms of control of growth of mycobacteria.

In conclusion, we demonstrate for the first time the molecular mechanism(s) involved in SP-A–mediated suppression of NO production by IFN-– and IFN- plus M. avium–stimulated murine AMs. SP-A decreases NF-B activation that is required for induction of both iNOS and TNF- genes. This study also highlights the importance of SP-A levels in the lung, as changes in SP-A levels may modulate the local lung defense system.

	Acknowledgments

 
This study was supported by NIH grants R01 HL61285 and R01 AI48455.

Received in original form May 20, 2002

Received in final form November 6, 2002

	References

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