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Published ahead of print on August 12, 2004, doi:10.1165/rcmb.2004-0003OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 31, pp. 587-594, 2004
© 2004 American Thoracic Society
DOI: 10.1165/rcmb.2004-0003OC

Accumulation of Inhibitory {kappa}B-{alpha} as a Mechanism Contributing to the Anti-Inflammatory Effects of Surfactant Protein–A

Yingda Wu, Stefanie Adam, Lutz Hamann, Holger Heine, Artur J. Ulmer, Ute Buwitt-Beckmann and Cordula Stamme

Department of Anesthesiology, First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou, China; Departments of Immunochemistry and Biochemical Microbiology, and of Immunology and Cell Biology, Research Center Borstel, Leibniz-Center for Medicine and Biosciences, Borstel; Institute of Microbiology and Hygiene, Charité, Humboldt University Berlin, Berlin; and Department of Anesthesiology, University of Lübeck, Lübeck, Germany

Address correspondence to: Cordula Stamme, M.D., Dept. of Immunochemistry and Biochemical Microbiology, Research Center Borstel, Parkallee 22, 23845 Borstel, Germany. E-mail: cstamme{at}fz-borstel.de


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The collectin surfactant protein (SP)-A has been implicated in multiple immunoregulatory functions of innate pulmonary host defense via modulating immune responses both in vitro and in vivo. The aim of the present study was to investigate mechanisms responsible for the anti-inflammatory effects of human (hu) SP-A on the inhibitory {kappa}B (I{kappa}B)/nuclear factor (NF)-{kappa}B signaling pathway in alveolar macrophages (AMs). Initial CD25 expression analysis by flow cytometry of CD14/hu Toll-like receptor 4–transfected Chinese hamster ovary reporter cells demonstrated that SP-A alone does not induce any NF-{kappa}B–dependent CD25 expression in these cells. In AMs, SP-A pretreatment caused a marked inhibition of lipopolysaccharide (LPS)-induced NF-{kappa}B activation independent of the LPS chemotype used as determined by electrophoretic mobility shift assay. Western blot analysis revealed that SP-A by itself increased the protein expression of I{kappa}B-{alpha}, the predominant regulator for rapidly induced NF-{kappa}B, in a dose- and time-dependent manner without enhancing I{kappa}B-{alpha} messenger RNA as determined by reverse transcription-polymerase chain reaction. SP-A did not interfere with LPS-induced serine32 phosphorylation of I{kappa}B-{alpha} but significantly enhanced I{kappa}B-{alpha} abundance under LPS-coupled conditions. The data suggest that anti-inflammatory effects of SP-A on LPS-challenged AMs are associated with a SP-A-mediated direct modulation of the I{kappa}B-{alpha} turnover in these cells.

Abbreviations: antibody, Ab • alveolar macrophages, AMs • Chinese hamster ovary, CHO • casein kinase II, CKII • electrophoretic mobility shift assay, EMSA • fetal calf serum, FCS • heat-inactivated, HI • horseradish peroxidase, HRP • human, hu • immunoglobulin, Ig • inhibitory {kappa}B, I{kappa}B • I{kappa}B kinase, IKK • interleukin, IL • lipopolysaccharide, LPS • nuclear factor, NF • proteasome inhibitor, PSI • reverse transcription–polymerase chain reaction, RT-PCR • rough LPS, R-LPS • smooth LPS, S-LPS • serine residue, Ser • surfactant protein A, SP-A • human monocytic leukemic cell line, THP-1 • Toll-like receptor, TLR • tumor necrosis factor, TNF


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Pulmonary surfactant protein (SP)-A is the most abundant surfactant protein and belongs to the collectin family of proteins along with, for example, SP-D, mannose-binding protein, and bovine conglutinin (1). The combination of a collagen-like region and a C-type lectin domain are the characteristic structural features of collectins which act as molecules of the innate immune system. The pulmonary collectins SP-A and SP-D are central modulators of the innate host defense and inflammation in the lung. SP-A binds and aggregates a variety of clinically relevant mircroorganisms, directly modulates the state of activation of macrophages, and enhances phagocytosis and killing of pathogens by immunocompetent cells (2, 3). SP-A–deficient mice have increased susceptibility to pulmonary infections with various bacterial and viral pathogens, and SP-A deficiency is associated with enhanced pulmonary inflammation (4).

The invasion of Gram-negative bacteria elicits immune responses, a pivotal mediator of which is lipopolysaccharide (LPS), an abundant component of the outer membrane of Gram-negative bacteria. Whereas LPS recognition benefits the host by sensing bacteria and mobilizing defense mechanisms, an exaggerated response to LPS contributes to the development of sepsis, septic shock, and death. Gram-negative bacteria express either smooth (S-) or rough (R-) LPS mutants. S-LPS is composed of O-antigen, complete core oligosaccharides, and lipid A. All mutant LPS chemotypes lack O-antigen, but possess lipid A and progressively shorter core oligosaccharides (5). LPS recognition by myeloid cells is initiated by Toll-like receptor (TLR)-4, LPS-binding protein, CD14, and MD-2 (6–8). Among the first cells to become activated after exposure to LPS are macrophages and monocytes. Upon LPS stimulation, these cells secrete proinflammatory mediators, such as tumor necrosis factor (TNF)-{alpha}, interleukin (IL)-6, IL-1ß, and reactive nitrogen species. The signal pathways required for this response include an increase in the DNA binding activity of NF-{kappa}B, a ubiquitous transcription factor that plays an essential role in the regulation of a broad variety of genes involved in immune and inflammatory reactions (8, 9). In resting cells, dimeric NF-{kappa}B is sequestered in the cytoplasm by a family of inhibitory proteins, collectively termed inhibitory {kappa}B (I{kappa}B). Although there are many different members of the I{kappa}B family, the best-characterized NF-{kappa}B activation pathway involves phosphorylation and degradation of the I{kappa}B-{alpha} isoform (10), which is assumed to function as the primary regulator of NF-{kappa}B in both stimulated and resting cells (11). In response to a variety of agents, including LPS, I{kappa}B-{alpha} is rapidly phosphorylated at two specific serine residues (Ser32 and Ser36) (10) and, subsequently, a substrate for ubiquitin ligases that catalyze the addition of ubiquitin groups to I{kappa}B-{alpha}, which targets I{kappa}B-{alpha} for degradation by the 26S proteasome (12). The removal of I{kappa}B-{alpha} allows the nuclear translocation of the thereby-activated NF-{kappa}B dimer. Degradation of I{kappa}B-{alpha} is followed by I{kappa}B-{alpha} mRNA induction through the binding of NF-{kappa}B to the {kappa}B promoter sequence of the I{kappa}B-{alpha} gene (13) to terminate the phase of NF-{kappa}B activation. This transcriptional autoregulatory loop is assumed to facilitate the re-emergence of I{kappa}B-{alpha} after activation.

There is currently considerable interest in the study of mechanisms that operate to regulate immunomodulatory effects of SP-A, both in the presence and absence of pro-inflammatory stimuli (14–19). Direct modulation of immunocompetent cells via interaction with SP-A has been implicated in the anti-inflammatory effects of SP-A. SP-A has been shown to inhibit TNF-{alpha} production (15, 18, 20), inducible nitric oxide synthase protein expression and nitrite production (21), and NF-{kappa}B activity (18, 19, 22) induced by S-LPS, which is not a ligand for SP-A (23). In addition, SP-A was found to inhibit NF-{kappa}B activation upon peptidoglycan, which is also not a ligand for SP-A, in human embryonic kidney 293 cells (24). Furthermore, proinflammatory cell activation induced by Candida and zymosan has been shown to be significantly inhibited by SP-A (25, 26). Importantly, SP-A–deficient mice demonstrated higher S-LPS–induced pulmonary cytokine and nitric oxide production than that observed in wild-type mice (27), and C57BL/6 mice injected intraperitoneally with SP-A before S-LPS challenge showed suppression of peritoneal cytokine release (18).

The data on the role of SP-A in immunomodulation are compelling, but the intracellular events by which SP-A exerts anti-inflammatory effects on LPS-activated immune cells are only partially understood. Because I{kappa}B-{alpha} is the primary regulator for rapidly induced NF-{kappa}B, the current study aimed to investigate the relationship between the anti-inflammatory effects of SP-A and the I{kappa}B-{alpha}/NF-{kappa}B signaling pathway in alveolar macrophages (AMs) under both basal and LPS signal–dependent conditions. Initial control experiments showed that SP-A alone does not induce NF-{kappa}B–dependent CD25 expression in CD14/huTLR4 Chinese hamster ovary (CHO) reporter cells. In AMs, SP-A pretreatment caused a marked inhibition of LPS-induced NF-{kappa}B DNA binding activity independent of the LPS chemotype used. SP-A by itself increased the protein expression of I{kappa}B-{alpha} in a dose- and time-dependent manner without enhancing I{kappa}B-{alpha} gene transcription. SP-A did not interfere with LPS-induced Ser32 phosphorylation of I{kappa}B-{alpha} but significantly enhanced I{kappa}B-{alpha} abundance under LPS-coupled conditions. The data suggest that direct anti-inflammatory effects of SP-A on LPS-stimulated macrophages are associated with an SP-A–mediated modulation of the I{kappa}B-{alpha} turnover in AMs.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents and Materials
The rough mutant LPS from Escherichia coli strain F515 was extracted by the phenol/chloroform/petroleum ether method (28), purified, lyophilized, and transformed into the triethylamine salt form. Synthetic lipopeptide (Pam3CysSerLys4) was purchased from EMC Microcollections (Tübingen, Germany). RPMI 1640 medium, Dulbecco's modified Eagle's medium, Dulbecco's phosphate-buffered saline, and TRIZOL reagent were from GIBCO BRL (Paisley, Scotland). Ham's-F12 medium and fetal calf serum (FCS) were from BioWhittaker (Verviers, Belgium). Nonspecific competitor DNA poly/deoxyinosine-deoxycytosine (dI/dC) was purchased from Pharmacia (Freiburg, Germany). [{gamma}-32P] ATP was supplied by Hartmann (Braunschweig, Germany), T4 polynucleotide kinase was purchased from Roche (Mannheim, Germany). Rabbit polyclonal anti–I{kappa}B-{alpha} antibody (Ab) and horseradish peroxidase (HRP)–conjugated goat anti-rabbit immunoglobulin (Ig) G were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Rabbit polyclonal anti–phosphorylated-I{kappa}B-{alpha} Ab (Ser32) was supplied by New England Biolabs (Beverly, MA). Mouse monoclonal anti-human phycoerythrin-CD25 Ab was obtained from Dako (Hamburg, Germany). Puromycin, hygromycin B, and the proteasome inhibitor (PSI) were purchased from Calbiochem (Bad Soden, Germany). All other chemicals (except as noted) were from Sigma (Deisenhofen, Germany).

Protein Purification
Human SP-A (generously provided by J. R. Wright, Department of Cell Biology, Duke University Medical Center, Durham, NC) was purified from the bronchoalveolar lavage fluid of patients with alveolar proteinosis as previously described in detail (20). SP-A was treated with polymyxin B agarose beads to reduce endotoxin contamination. SP-A preparations were tested for the presence of bacterial endotoxin using the Limulus amebocyte lysate assay (Bio-Whittaker, Walkersville, MD); all SP-A preparations used contained < 0.2 pg endotoxin per µg SP-A.

Isolation of Cells and Incubation Conditions
AMs were isolated by lung lavage of male Sprague-Dawley rats weighing 200–250 g (Charles River, Sulzfeld, Germany) as previously described (21). Cell recovery routinely averaged 4–8 x 106 cells/animal. The viability of the cells was determined by erythrosin B exclusion and averaged 94–98%. Cells were plated at 0.8 x 106/500 µl in 24-well plates (Costar, Bodenheim, Germany) and allowed to attach for 2 h. The medium was then changed and the cells were treated with SP-A (5–60 µg/ml) for indicated times at 37°C in the presence of 0.2% heat-inactivated (HI) FCS and, for some experiments, subsequently stimulated with R-LPS (E. coli F515) or S-LPS (E. coli 0111:B4) (10 ng/ml) for indicated times (Figures 4 and 6). In separate experiments, cells were pretreated with the proteasome inhibitor PSI (15 µM) for 1 h.

Cell Lines and Flow Cytometry Analysis of NF-{kappa}B Activity
The CHO/CD14 reporter line, clone 3E10, is a stably transfected CD14-positive CHO cell line that expresses inducible membrane CD25 (Tac Ag; {alpha}-chain of the IL-2R) under transcriptional control of an NF-{kappa}B–dependent fragment of the human E-selectin promoter (29). LPS, TNF{alpha}, and IL-1 all activate NF-{kappa}B in these cells, resulting in a 3- to 10-fold increase in the surface expression of CD25. The CHO/CD14/huTLR2 reporter cell line was constructed by stable cotransfection of 3E10 with the cDNA for human TLR2 and pcDNA3 (Invitrogen, CA), as previously described (30). The CHO/CD14/huTLR4 reporter cell line was generated in the same manner, except that a puromycin resistance plasmid (pRc/RSV; a generous gift of R. Kitchens, University of Texas, Southwestern Medical Center, Dallas, TX) was used for drug selection (31). CHO cell lines were grown in Ham's F12 medium containing 10% FCS and 1% penicillin/streptomycin at 37°C in a humidified 5% CO2 environment. Medium was supplemented with 400 U/ml of hygromycin B and 0.5 mg/ml of G418 (CHO/CD14/huTLR2) or 50 µg/ml of puromycin (CHO/CD14/huTLR4). Cells were plated at a density of 2.5 x 105/well in 24-well dishes. The following day, the cells were stimulated as indicated in Ham's F12 medium containing 10% FCS (total volume of 0.3 ml/well) (Figure 1). Subsequently, the cells were harvested with trypsin–ethylenediamine tetraacetic acid, labeled with mouse anti-human phycoerythrin-CD25 monoclonal Ab and analyzed by flow cytometry (FACS Calibur; Becton Dickinson, Heidelberg, Germany). Binding of anti-CD25 Ab to its epitope is expressed as percentage of IL-1–induced activation.



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  		Figure 1. SP-A does not activate NF-{kappa}B in CHO/CD14/huTLR4 reporter cell lines. CD25 expression analysis was performed after stimulation of CHO/CD14/huTLR4 reporter cell lines with LPS (10–1,000 ng/ml), and SP-A (1–10 µg/ml). Cells were plated at a density of 2.5 x 105/well in 24-well dishes. The following day, the cells were stimulated as indicated for 20 h. Subsequently, the cells were harvested with trypsin–ethylenediamine tetraacetic acid, and expression of CD25 was analyzed by flow cytometry. *CD25 expression induced by IL-1 (25 U/ml) in each cell line has been set to 100%. Gray bars, CHO/CD14; black bars, CHO/CD14/huTLR4.

 
Nuclear Protein Extraction and NF-{kappa}B Activation Assay
After exposing cells to the experimental conditions, nuclear extracts were prepared and analyzed as described previously (17). The cells were scraped off the plates, centrifuged, and resuspended in 400 µl of ice-cold buffer A (10 mM Tris, 5 mM MgCl2, 10 mM KCl, 1 mM ethyleneglycol-bis-(2-aminoethyl ether)-N,N,N',N'-tetraacetic acid, 0.3 M sucrose, 1 mM dithiothreitol, 0.5 mM phenylmethylsulfonylfluoride, 10 mM ß-glycerol phosphate, and 1.5 µl of protease inhibitor cocktail (Complete; Roche). After 15 min on ice, 25 µl of 10% NP-P40 was added. The solution was vortexed and nuclei were pelleted by centrifugation. The supernatants (cytoplasmic extracts) were collected and snap frozen at –80°C. The nuclear pellet was resuspended in 30 µl of buffer B (20 mM Tris, 5 mM MgCl2, 320 mM KCl, 0.2 mM ethyleneglycol-bis-(2-aminoethyl ether)-N,N,N',N'-tetraacetic acid, 1 mM dithiothreitol, 25% glycerol, and the mixture of protease inhibitors mentioned above) and, after 15 min on ice, lysates were cleared by centrifugation at 14,000 x g for 15 min. The activity of NF-{kappa}B in the nuclear extracts was determined by a standard electrophoretic mobility shift assay (EMSA). NF-{kappa}B oligonucleotides were end-labeled with [{gamma}-32P] ATP using T4 kinase. A 2-µg sample of crude nuclear extract was incubated for 20 min in binding buffer containing 50 µg of poly (dI/dC)/ml with 7.5 fmol of the 32P-labeled oligonucleotide encoding the consensus NF-{kappa}B site 5'-AGCTCAGAGGGGACTTTCCGAGAGAGC-3' (MWG-Biotech, Ebersberg, Germany). Samples were separated by electrophoresis in 5% polyacrylamid gels for 2 h at 150 V, after which gels were analyzed with a PhosphorImager (Molecular Dynamics, Krefeld, Germany). In competition experiments, 100x unlabeled NF-{kappa}B probe was added along with radiolabeled NF-{kappa}B probe. For supershift analysis, nuclear extracts were incubated with anti-p65 and anti-p50 antibodies for 20 min before addition of radiolabeled probe.

Western Blot Analysis
To determine if SP-A affects I{kappa}B-{alpha} protein expression in AMs, Western blot analysis was performed. After treatment, whole-cell lysates, or cytosolic and nuclear fractions, were assayed for protein content by the bicinchoninic acid reagent (Pierce, Rockford, Il, USA). Proteins of 20–30 µg were resolved on sodium dodecyl sulphate–polyacrylamide gel electrophoresis (12%) and transferred to nitrocellulose membrane. Nonspecific binding sites were blocked with 5% nonfat dry milk in 10 mM Tris, 100 mM NaCl, and 0.1% Tween 20 for 1 h at room temperature. The membranes were then incubated in 0.1% Tween 20 with 5% nonfat dry milk with rabbit polyclonal anti–I{kappa}B-{alpha} Ab or rabbit monoclonal anti–phosphorylated I{kappa}B-{alpha} at a 1:700 and 1:200 dilution, respectively. Goat anti-rabbit IgG–HRP served as the secondary antibody. Immunoreactive proteins were visualized by enhanced chemiluminescence using the ECL Western blotting detection system (Amersham Pharmacia Biotech, Buckinghamshire, UK). Samples were also assessed for actin content as an internal control. Membranes were incubated with a mouse monoclonal anti-actin IgG (Chemicon, Hofheim, Germany) at a 1: 2,000 dilution. Rabbit anti-mouse IgG–HRP at a 1: 5,000 dilution served as the secondary antibody.

Reverse Transcription–Polymerase Chain Reaction Analysis
Total cellular RNA was isolated from the cells using TRIzol reagent, and 1 µg of RNA was reverse transcribed with oligo(dT) primers. Polymerase chain reaction (PCR) was performed with 1 U of Taq polymerase (Bioline, Luckenwalde, Germany) in a total volume of 50 µl. The primers (MWG-Biotech AG) used for I{kappa}B-{alpha} were 5'-CATGAAGAGAAGACACTGACCATGGAA-3' and 5'-TGGATAGAGGCTAAGTGTAG ACACG-3', which yield a 328-bp product. Primers used for glyceraldehyde-3-phosphate dehydrogenase detection were 796F (5'-GCCAAGTATGATGACATCAAGAAG) and 1059R (5'-TCCAGGGGTTTCTTACTCCTTGGA), yielding a 264-bp product. PCR conditions were 34 cycles of denaturation at 93°C for 15 s, annealing at 63°C for 20 s, and extension at 72°C for 20 s followed by 5 min at 72°C in a Biometra UNO II Thermoreactor (Biometra, Göttingen, Germany). PCR products were separated by electrophoresis through 1% agarose gels containing 0.1 µg/ml ethidium bromide. The identity of the PCR products was confirmed by direct sequencing (ABI 3100; Applied Biosystems, Foster City, CA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The Expression of Human TLR4 in CD14/CHO Reporter Cells Does Not Confer Responsiveness to SP-A
Several laboratories reported that SP-A alone has no proinflammatory effect on immunocompetent cells (15, 17–22, 24–26). Conflicting reports have been published showing that SP-A itself could induce NF-{kappa}B activity in human monocytic THP-1 cells (14). This proinflammatory effect of SP-A has been proposed to be similar to that of LPS (e.g., via CD14 and TLR4 complex–mediated signal transduction pathways) (16). In the current study, NF-{kappa}B–dependent CD25 expression analysis of CD14/CHO reporter cells demonstrated by flow cytometry that the expression of huTLR4 does not confer responsiveness to SP-A (1–10 µg/ml) compared with LPS (10–1,000 ng/ml) (Figure 1). SP-A has been shown to bind to TLR2 (24, 26), which recognizes and mediates signals for lipoproteins, lipopeptides, and peptidoglycan. In the current investigation, SP-A did not activate CD14/CHO/huTLR2 cells, whereas the synthetic lipopeptide Pam3CysSerLys4 induced a dose-dependent increase of CD25 expression, reaching a maximum of 68% of control at a concentration of 1,000 nM.

SP-A Inhibits R- and S-LPS–Induced NF-{kappa}B Activity in AMs
The activation effect of NF-{kappa}B in AMs upon 10 ng/ml R-LPS was apparent after 30 min, reached a maximum after 60 min, and then declined (Figure 2A). The time-course of NF-{kappa}B activation induced by S-LPS (10 ng/ml) was comparable (data not shown). We initiated this study by extending the analysis of the ability of SP-A to directly inhibit the activation of AMs by pretreatment of the cells with SP-A and the use of distinct LPS chemotypes (i.e., S-LPS, to which SP-A does not bind, and R-LPS, to which SP-A avidly binds). The treatment of AMs with SP-A did not induce TNF-{alpha} release by these cells, but significantly inhibited R-LPS and S-LPS–induced TNF-{alpha} release (data not shown). The EMSA results showed that SP-A alone has no effect on NF-{kappa}B activity compared with LPS stimulation, but the preincubation of AMs with SP-A for 2 h inhibits NF-{kappa}B activation induced by both R-LPS (10 ng/ml) and S-LPS (10 ng/ml) by 43% and 45%, respectively (Figure 2C). To investigate whether the presence of SP-A in the medium is required to inhibit NF-{kappa}B activity, AMs were incubated with SP-A for 1 h and the cells were washed and then stimulated with LPS for 1 h. The EMSA results indicate that cell-bound SP-A reduced NF-{kappa}B activity in S-LPS– (37%) and R-LPS– (42%) stimulated AMs. Excess unlabeled NF-{kappa}B caused complete disappearance of the band, indicating the specifity of NF-{kappa}B. Supershift analysis experiments confirmed the composition of the NF-{kappa}B/DNA complex to contain RelA (p65) (Figure 2B).



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  		Figure 2. SP-A inhibits LPS-induced NF-{kappa}B activity in AMs independent of the LPS chemotype used. (A) Kinetic of R-LPS–induced NF-{kappa}B activation in AMs. AMs were stimulated with R-LPS (10 ng/ml) for the times indicated. (B) Treatments in each lane were control (1), LPS (2), LPS plus 100-fold excess of unlabeled oligonucleotides (3), LPS plus anti-p50 antibodies (4), LPS plus anti-p65 antibodies (5), and LPS plus nonspecific unlabeled oligonucleotides (AP-1) (6). (C) AMs (0.8 x 106/well) were preincubated in the absence or presence of SP-A (5 µg/ml) for 2 h at 37°C, followed by the addition of R-LPS (10 ng/ml) or S-LPS (10 ng/ml) to some samples for 1 h in the presence of 0.2% HI-FCS. Samples were assayed for NF-{kappa}B translocation by EMSA as described in MATERIALS AND METHODS. Data illustrated are from single experiments and are representative of three to five experiments.

 
SP-A Enhances Basal Cytosolic I{kappa}B-{alpha} Protein Levels
Because NF-{kappa}B is retained in the cytoplasm by its interaction with I{kappa}B-{alpha}, and the degradation of I{kappa}B-{alpha} is a prerequisite for the activation of NF-{kappa}B, we first tested whether SP-A by itself affects the cytosolic I{kappa}B-{alpha} protein level. As shown in Figure 3, SP-A caused a dose-dependent increase in I{kappa}B-{alpha} levels in AMs incubated for 90 min in the presence of 0.2% HI-FCS. Maximal enhancement was observed in the presence of 60 µg/ml of SP-A, which resulted in an increase in I{kappa}B-{alpha} protein expression by ~ 250% over control (no SP-A) as assessed by densitometric analysis (Figure 3B). In the absence of serum, the SP-A–mediated enhancement of I{kappa}B-{alpha} protein was 143 ± 23% (n = 4, P < 0.05) at 10 µg/ml of SP-A and 171 ± 36% (n = 4, P < 0.05) at 60 µg/ml of SP-A after 60 min of incubation. Time-course experiments revealed that the incubation of cells with SP-A (40 µg/ml) resulted in a 273 ± 34%, 301 ± 48%, and 494 ± 119% enhancement of I{kappa}B-{alpha} protein at 10, 60, and 120 min, respectively, suggesting that the effect of SP-A is increasing over time (Figure 4B). In contrast with SP-A, neither C1q nor heat-inactivated SP-A affected cytosolic I{kappa}B-{alpha} protein levels (data not shown).



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  		Figure 3. SP-A in a dose-dependent manner enhances basal cytosolic I{kappa}B-{alpha} protein levels. (A) AMs (0.8 x 106/well) were incubated with SP-A (0, 20, and 60 µg/ml) or R-LPS (10 ng/ml) in the presence of 0.2% HI-FCS for 90 min at 37°C in a 5% CO2 atmosphere. Equal amounts of cytosolic fractions (20 µg) were subjected to Western blot analysis for I{kappa}B-{alpha} expression. (B) Dose-dependent effects of SP-A on I{kappa}B-{alpha} protein expression. AMs (0.8 x 106/well) were incubated with SP-A (5, 20, 40, and 60 µg/ml) in the presence of 0.2% HI-FCS for 90 min at 37°C in a 5% CO2 atmosphere. Equal amounts of cytosolic fractions (20 µg) were subjected to Western blot analysis for I{kappa}B-{alpha} expression. Results from three to five separate experiments were analyzed by using OPTIMAS 6.2 software (Optimas Corp., Bothell, WA) and presented as a percentage of I{kappa}B-{alpha} in the absence of SP-A (control, 100%). *P < 0.01 compared with control.

 


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  		Figure 4. SP-A enhances basal cytosolic I{kappa}B-{alpha} protein levels in a time-dependent manner. (A) AMs (0.8 x 106/well) were incubated with SP-A (40 µg) in the presence of 0.2% HI-FCS for the times indicated at 37°C in a 5% CO2 atmosphere. Equal amounts of cytosolic fractions (20 µg) were subjected to Western blot analysis for I{kappa}B-{alpha} expression. (B) Time-dependent effects of SP-A on I{kappa}B-{alpha} protein expression. Results from five to seven separate experiments were analyzed by using OPTIMAS 6.2 software (Optimas Corp.) and presented as a percentage of I{kappa}B-{alpha} at the earliest time point (~ 20 s) in the presence of SP-A. *P < 0.05 compared with control.

 
SP-A Does Not Enhance I{kappa}B-{alpha} mRNA Expression
To determine if the SP-A–mediated increase in I{kappa}B-{alpha} protein was preceded by an increase in I{kappa}B-{alpha} mRNA, RT-PCR was performed on mRNA from AMs incubated with SP-A for increasing periods of time. However, within the timeframe investigated, in contrast with controls, SP-A did not affect I{kappa}B-{alpha} mRNA expression (Figure 5).



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  		Figure 5. SP-A does not enhance I{kappa}B-{alpha} mRNA expression. AMs (0.8 x 106/well) were incubated in the absence (lanes 1, 2, 3, and 4) or presence of SP-A (40 µg/ml) (lanes 5, 6, 7, and 8) for 10, 30, 60, and 90 min. RNA was isolated and I{kappa}B-{alpha} and glyceraldehyde-3-phosphate dehydrogenase mRNA were estimated by RT-PCR as described in MATERIALS AND METHODS. Data shown are representative of three independent experiments.

 
SP-A Inhibits LPS-Induced I{kappa}B-{alpha} Degradation
Next we tested whether the inhibitory effect of SP-A on LPS-induced NF-{kappa}B activation was associated with its ability to modulate cytosolic and nuclear I{kappa}B-{alpha} abundance. Cytosolic I{kappa}B-{alpha} protein levels from control (no SP-A) and SP-A– (40 µg/ml) pretreated cells after stimulation with R- and S-LPS (10 ng/ml) for 10 to 60 min were determined by Western blot analysis. As expected, LPS-stimulated AMs exhibited a rapid degradation of I{kappa}B-{alpha} within 20 to 40 min, with a reappearance of the protein at 60 min. As shown in Figures 6A and 6B, pretreatment of the cells with SP-A markedly enhanced I{kappa}B-{alpha} content under LPS-coupled conditions. After 20 min of LPS treatment, at which time maximum I{kappa}B-{alpha} degradation was observed, SP-A pretreatment for 2 h reduced the degradation of I{kappa}B-{alpha} by 2.6-fold. To further investigate the time-course of SP-A–mediated inhibition of LPS-induced I{kappa}B-{alpha} degradation, AMs were pretreated with SP-A for a variable period of time before LPS administration. Pretreatment of the cells with SP-A for 10 min inhibited LPS-induced I{kappa}B-{alpha} degradation at 20 min by 1.9-fold (mean from two different experiments). Increases in I{kappa}B-{alpha} protein levels were detected in both the cytosolic and the nuclear fraction (Figure 6C), suggesting that SP-A also influences the subcellular localization of I{kappa}B-{alpha}.



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  		Figure 6. SP-A enhances I{kappa}B-{alpha} abundance under LPS-coupled conditions. AMs (0.8 x 10 6/well) were preincubated in the absence or presence of SP-A (40 µg/ml) in the presence of 0.2% HI-FCS for 2 h at 37°C in a 5% CO2 atmosphere, followed by the addition of (A) R-LPS (10 ng/ml) or (B) S-LPS (10 ng/ml) for the indicated times. Equal amounts of cytosolic fractions (25 µg) were subjected to Western blot analysis for I{kappa}B-{alpha} expression. Data illustrated are from a single experiment and are representative of at least three experiments. (C) AMs were treated as in (B). Equal amounts of nuclear and cytosolic extracts (15 µg) were subjected to sodium dodecyl sulphate–polyacrylamide gel electrophoresis, and transferred proteins were analyzed by Western blot for the presence of I{kappa}B-{alpha}. C, cytosolic extracts; N, nuclear extracts. Data shown are representative out of two independent experiments.

 
LPS-Induced I{kappa}B-{alpha} Phosphorylation at Ser32 Is Not Affected by SP-A
Because LPS-induced I{kappa}B-{alpha} degradation is preceded by I{kappa}B kinase (IKK)–dependent phosphorylation of two serine residues within the N terminus (Ser32 and Ser36) (10), we determined the LPS-induced phosphorylation of I{kappa}B-{alpha} from control (no SP-A) and SP-A–pretreated cells to evaluate a possible mechanism of the SP-A–mediated enhancement of I{kappa}B-{alpha} levels. To stabilize the labile phosphorylated I{kappa}B-{alpha}, control (no SP-A) and SP-A–pretreated cells were incubated with the relatively specific proteasome inhibitor PSI (15 µM) for 60 min before the addition of LPS. Whole-cell lysates were then subjected to Western blot analysis. As shown in Figure 7, incubation of the cells with PSI/LPS resulted in enhanced phosphorylation of I{kappa}B-{alpha} in contrast with LPS alone (10 ng/ml), whereas SP-A alone had no effect on I{kappa}B-{alpha} phosphorylation. In addition, SP-A did not inhibit LPS-induced Ser32 phosphorylation of I{kappa}B-{alpha}, suggesting that SP-A does not interfere with IKK-dependent LPS signal–induced phosphorylation of I{kappa}B-{alpha}.



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  		Figure 7. SP-A does not affect Ser32 phosphorylation of I{kappa}B-{alpha}. AMs (0.8 x 106/well) were preincubated in the absence or presence of SP-A (40 µg/ml) with 1% HI-FCS for 2 h at 37°C in a 5% CO2 atmosphere. In separate experiments, cells were pretreated with the proteasome inhibitor PSI (15 µM) for 1 h. The cells were then challenged with R-LPS (10 ng/ml) for 40 min. Western blot analysis was performed with whole-cell extracts using an antibody specific to phosphorylated I{kappa}B-{alpha} on Ser32. Data illustrated are from a single experiment and are representative of at least three experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the current study, we investigated mechanisms responsible for the anti-inflammatory action of SP-A on NF-{kappa}B DNA binding activity. Because I{kappa}B-{alpha} is assumed to be the primary regulator of NF-{kappa}B in both unstimulated and stimulated cells (11), the relationship between direct anti-inflammatory effects of SP-A and the I{kappa}B-{alpha}/NF-{kappa}B signaling pathway in AMs was investigated. Initial CD25 expression analysis of CD14/CHO/huTLR4 cell lines showed that huTLR4 expression does not confer responsiveness to the SP-A preparation used. In freshly isolated alveolar macrophages, SP-A inhibited NF-{kappa}B activation by R-LPS, to which it binds, and S-LPS, to which it does not bind (23), suggesting a direct anti-inflammatory effect of SP-A on these cells. SP-A by itself increased cytosolic I{kappa}B-{alpha} protein levels in a dose- and time-dependent manner without enhancing I{kappa}B-{alpha} gene transcription. SP-A did not interfere with LPS-induced Ser32 phosphorylation of I{kappa}B-{alpha} but markedly enhanced I{kappa}B-{alpha} content under LPS-coupled conditions.

CD25 expression analysis of CHO reporter cells showed that SP-A does not induce any NF-{kappa}B activation in CHO/CD14/TLR4 cell lines, suggesting that the SP-A preparations used have no stimulatory effects on the LPS signaling pathway. Interestingly, a recent study by Guillot and coworkers (16) demonstrated that SP-A alone was efficient in activating the NF-{kappa}B signaling pathway in these cells. These authors showed that the pro-inflammatory effects of SP-A were resistant to polymyxin B treatment. In contrast, we did not observe any activation of NF-{kappa}B using polymyxin-purified SP-A. However, in the study by Guillot and coworkers, SP-A was purified by different methods than those employed in our study, and this difference may have had an impact on immunogenicity. SP-A was shown to bind to a soluble form of the recombinant extracellular TLR2 domain without inducing NF-{kappa}B activation in human embryonic kidney 293 cells transiently transfected with TLR2 (24, 26). In line with this observation, we found that SP-A did not induce NF-{kappa}B–dependent CD25 expression in CHO/CD14/TLR2 cells.

NF-{kappa}B has a pivotal role in controlling both innate and adaptive immunity. Because the transcription of most pro-inflammatory genes, including the TNF-{alpha} gene, is dependent on NF-{kappa}B activation (32), it is very likely that the suppression of TNF-{alpha} by SP-A is closely related to the blocking of NF-{kappa}B activation. Our EMSA data demonstrate that SP-A alone does not activate NF-{kappa}B in freshly isolated AMs, but significantly inhibits LPS-induced NF-{kappa}B activation by these cells independent of the LPS chemotype used. In contrast, it was shown that SP-A increases NF-{kappa}B activity in vitamin D3-differentiated THP-1 cells (14). Because vitamin D3 treatment of THP-1 cells modulates their sensitivity (33), the contradictory results may depend largely on both cell specificity and experimental design. Our findings are consistent with several recent reports demonstrating inhibitory effects of SP-A on LPS-induced NF-{kappa}B activation in alveolar macrophages (17–19, 22). Importantly, the mechanisms involved have been shown to be both dependent and independent of the LPS signaling pathway. Traditionally, NF-{kappa}B activity has been considered to be differentially regulated through I{kappa}B-{alpha} phosphorylation and NF-{kappa}B–dependent resynthesis of I{kappa}B-{alpha} (13, 34). In the current study, we first tested the possibility that SP-A alone affects cytosolic I{kappa}B-{alpha} protein levels in resting AMs. The data show that, under resting conditions, SP-A markedly enhances the cytosolic I{kappa}B-{alpha} protein level in a dose- and time-dependent manner. Importantly, the SP-A concentrations used were within the predicted physiologic range (2). The effect was significant in the complete absence of serum, suggesting that no serum-derived protein is required for the SP-A–mediated increase in I{kappa}B-{alpha}. Because the I{kappa}B-{alpha} promoter is a canonical NF-{kappa}B–dependent gene (34), and we and others (17–19, 22, 24, 26, 35, and this study) have failed to document any SP-A–induced NF-{kappa}B activity, we proposed that SP-A acts via posttranscriptional mechanism(s) to enhance I{kappa}B-{alpha} protein abundance. Our RT-PCR experiments revealed that SP-A does not regulate I{kappa}B-{alpha} at the transcriptional level, arguing against de novo induction of I{kappa}B-{alpha} as a potential mechanism by which SP-A antagonizes NF-{kappa}B. Little is known about the mechanisms that regulate basal I{kappa}B-{alpha}/NF-{kappa}B activity. The regulation of I{kappa}B-{alpha} is performed mainly through phosphorylation. It has been shown that constitutive carboxyl-terminal casein kinase II (CKII) phosphorylation sites are necessary for the regulation of signal-independent turnover of I{kappa}B-{alpha} (36), and that I{kappa}B-{alpha} undergoes signal-independent ubiquitination (11). Interestingly, it has been shown that CKII-controlled p65 phosphorylation and NF-{kappa}B transactivation potential is modulated by I{kappa}B-{alpha} availability (37). SP-A has recently been found to modulate CKII activity in alveolar macrophages (38). Whether CKII modulation by SP-A plays a role in the basal I{kappa}B-{alpha} turnover and/or in the posttranslational modification of NF-{kappa}B proteins remains to be determined. Alternatively, SP-A has been shown to trigger a rapid tyrosine phosphorylation of a variety of proteins in AMs (39), and tyrosine phosphorylation of I{kappa}B-{alpha} can regulate NF{kappa}-B activity (40–42). In addition, SP-A was found to increase both phosphorylation and phosphatase activity of Src homology 2 domain-containing protein-tyrosine phosphatase 1 (SHP-1) (18), a soluble tyrosine phosphatase that participates in the negative regulation of the receptor tyrosine kinase pathway. SHP-1 has been shown to play a critical role in the transcriptional regulation of proinflammatory genes through the control of I{kappa}B protein stability (43). Studies are currently in progress to determine the specific phosphorylation sites and corresponding protein kinases/phosphatases involved in the regulation of I{kappa}B-{alpha} in resting and LPS-stimulated AMs exposed to SP-A.

Consistent with other studies (44, 45), we found that LPS-stimulated AMs exhibit a rapid degradation of cytosolic I{kappa}B-{alpha}, with a reappearance of the protein at ~ 60 min. Pretreatment of AMs with SP-A inhibited LPS-induced I{kappa}B-{alpha} degradation, although SP-A did not prevent Ser32 phosphorylation of I{kappa}B-{alpha}, suggesting that SP-A does not interfere with stimulus-induced IKK-dependent I{kappa}B-{alpha} phosphorylation. The data suggest that SP-A directly inhibits LPS-induced NF-{kappa}B activation in AMs by providing a greater abundance of I{kappa}B-{alpha}. Because we observed that SP-A increases both cytosolic and nuclear levels of I{kappa}B-{alpha}, one might speculate that both cytosolic sequestration of NF-{kappa}B and the inhibition of NF-{kappa}B binding to specific DNA sequences are involved in the inhibitory effects on NF-{kappa}B activation. Because proteasome-mediated degradation is a principal factor controlling the intracellular levels of I{kappa}B-{alpha}, it would appear that SP-A is exerting its effect by inhibiting, directly or indirectly, the ubiquitin-proteasome pathway. Possible mechanisms include the inhibition of I{kappa}B-{alpha} polyubiquitylation and the inhibition of ubiquitylated I{kappa}B-{alpha} degradation by the 26S proteasome. Both mechanims have been shown to be involved in the anti-inflammatory effects of antibacterial peptide PR39 (46), secretory leucoprotease inhibitor (47), heat shock protein 70 (48), and Murr1 (49).

In conclusion, the data presented here suggest that SP-A has no cell-activating potency via CD14 and TLR4 complex–mediated signal transduction pathways, but exerts its anti-inflammatory effects on LPS-challenged AMs via a mechanism involving an SP-A–mediated direct modulation of the basal- and LPS-coupled I{kappa}B-{alpha} turnover in these cells. These data lend further credence to a physiologic role of SP-A in regulating pulmonary host defense and suggest a function of SP-A in altering the signaling threshold for NF-{kappa}B activation under resting and inflammatory conditions.


   Acknowledgments
 
The authors are grateful to Professor Jo Rae Wright, who supplied the human SP-A preparations. They thank Dr. Elvira Richter for sequencing of RT-PCR products. The technical assistance of Ina Goroncy and Katrin Klopfenstein is gratefully acknowledged. This work was supported by the Deutsche Forschungsgemeinschaft 609/1-1 and 1-2 (to C.S.) and the European Respiratory Society Fellowships 2002-006 and 2003-010 (to Y.W.).


   Footnotes
 
Conflict of Interest Statement: Y.W. has no declared conflicts of interest; S.A. has no declared conflicts of interest; L.H. has no declared conflicts of interest; H.H. has no declared conflicts of interest; A.J.U. has no declared conflicts of interest; U.B.-B. has no declared conflicts of interest; and C.S. has no declared conflicts of interest.

Received in original form January 4, 2004

Received in final form August 5, 2004


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
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