Surfactant protein A enhances mycobacterial killing by rat macrophages through a nitric oxide-dependent pathway

Laura F. Weikert1, Joseph P. Lopez2, Rasul Abdolrasulnia1, Zissis C. Chroneos3, and Virginia L. Shepherd2,4

Departments of 1 Medicine and 2 Pathology, Vanderbilt University School of Medicine, and 4 Veterans Affairs Medical Center, Nashville, Tennessee 37212; and 3 Children's Hospital Medical Center, Cincinnati, Ohio 45229


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Surfactant-associated protein A (SP-A) is involved in surfactant homeostasis and host defense in the lung. We have previously demonstrated that SP-A specifically binds to and enhances the ingestion of bacillus Calmette-Guerin (BCG) organisms by macrophages. In the current study, we investigated the effect of SP-A on the generation of inflammatory mediators induced by BCG and the subsequent fate of ingested BCG organisms. Rat macrophages were incubated with BCG in the presence and absence of SP-A. Noningested BCG organisms were removed, and the release of tumor necrosis factor-alpha (TNF-alpha ) and nitric oxide were measured at varying times. TNF-alpha and nitric oxide production induced by BCG were enhanced by SP-A. In addition, SP-A enhanced the BCG-induced increase in the level of inducible nitric oxide synthase protein. Addition of antibodies directed against SPR210, a specific macrophage SP-A receptor, inhibited the SP-A-enhanced mediator production. BCG in the absence of SP-A showed increased growth over a 5-day period, whereas inclusion of SP-A dramatically inhibited BCG growth. Inhibition of nitric oxide production blocked BCG killing in the presence and absence of SP-A. These results demonstrate that ingestion of SP-A-BCG complexes by rat macrophages leads to production of inflammatory mediators and increased mycobacterial killing.

phagocytosis; mycobacteria; surfactant-associated protein A


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

SURFACTANT-ASSOCIATED protein A (SP-A) is a member of the C-type lectin family and is synthesized and secreted by type II epithelial cells in the lung (44). The SP-A monomer has a molecular mass of 36 kDa, which forms 18-mers of greater than 600 kDa in size. The monomer contains an NH2-terminal collagen-like domain, which has been implicated in interactions with cell surface receptors (7, 29), and a COOH-terminal carbohydrate recognition domain, which appears to be involved in binding to a variety of microorganisms (44). It has been hypothesized that SP-A contributes to phospholipid homeostasis in the lung as well as participates in host defense against pulmonary pathogens (44). In vitro studies have shown that SP-A functions as an opsonin and enhances the ingestion of a number of pathogens, including bacillus Calmette-Guerin (BCG) (42), Mycobacterium tuberculosis (11), influenza A virus (2), Escherichia coli (32), Hemophilus influenzae (27, 37), Staphylococcus aureus (26), Streptococcus pneumoniae (37), Mycoplasma pulmonis (16), and Klebsiella pneumoniae (17). The importance of SP-A in in vivo host defense has been supported recently by the demonstration that mice deficient in SP-A show decreased resistance to group B streptococcal and Pseudomonas aeruginosa pneumonia (20, 22) and that SP-A enhances clearance of respiratory syncytial virus (RSV) (21). In in vitro studies, Kabha et al. (17) and Hickman-Davis et al. (16) have demonstrated recently that SP-A enhances the ingestion and killing of K. pneumoniae and mycoplasma by macrophages.

Alveolar macrophages are thought to have a critical role in host defense in the lung. Recent studies have suggested that SP-A plays an important role in the modulation of the inflammatory and immune responses of alveolar macrophages. SP-A promotes microbicidal activity of macrophages by enhancing the phagocytosis of microorganisms (14, 17, 40), by increasing the production of reactive oxygen species (43), and by stimulating macrophage chemotaxis (45). In certain systems, SP-A induces tumor necrosis factor-alpha (TNF-alpha ) and nitric oxide (3, 19), and activates nuclear factor-kappa B (NF-kappa B) (18). In addition, Borron et al. (4) have reported recently that SP-A blocks the costimulatory signals involved in T cell activation. These effects of SP-A on macrophages appear to be mediated through specific cell surface receptors (4, 29, 42). We have recently reported that macrophages express a receptor that binds SP-A and SP-A-mycobacteria complexes (6). This receptor (SPR210) is a 210-kDa membrane protein expressed on human monocytes, rat macrophages, and rat epithelial type II cells. Previous studies have shown that SPR210 mediates ingestion of SP-A-BCG complexes by macrophages (42) and mediates the effects of SP-A on T cells (4). However, the role that SPR210 plays in the ultimate fate of internalized pathogens is not known.

In the current study, we have extended our studies on the role that SP-A and SPR210 have in mycobacteria-macrophage interactions. Previous work has demonstrated that SP-A enhances attachment of M. tuberculosis to human alveolar macrophages (30) and that SP-A binds to BCG and enhances its ingestion by rat alveolar macrophages in vitro (42). Once inside the macrophage, survival of the mycobacteria is dependent on its escaping the bactericidal mechanisms of the host. In rodent systems, macrophages kill ingested mycobacteria through the induction of inducible nitric oxide synthase (iNOS) and subsequent production of nitric oxide (28). Interference in this process by iNOS inhibitors or the inability to produce nitric oxide in iNOS-deficient mice leads to mycobacterial growth and disease (24). In the current study, we have examined the effect of SP-A on BCG-induced production of inflammatory mediators and the role of these mediators in subsequent mycobacterial survival.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
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Materials. [5,6-3H]uracil was purchased from NEN (Boston, MA). Rat interferon-gamma (IFN-gamma ) was purchased from GIBCO BRL (Grand Island, NY). Fetal bovine serum for culture of rat bone marrow macrophages (RBMM) was purchased from HyClone Laboratories; all other tissue culture reagents were from GIBCO BRL. Neutralizing TNF-alpha antibody was purchased from GIBCO BRL. iNOS antibodies were purchased from Transduction Laboratories (Lexington, KY). All other reagents, including N-monomethyl-L-arginine (L-NMMA) and polymyxin B-agarose, were from Sigma (St. Louis, MO).

Purification of SP-A. SP-A was isolated from human alveolar proteinosis fluid (APF) by butanol extraction as previously described (7). The purified SP-A as analyzed by SDS-PAGE analysis showed only the monomer and dimer forms found in SP-A purified from APF. Before addition to cells, the SP-A preparations were treated with polymyxin to remove any contaminating lipopolysaccharide (LPS) as follows: 1 ml of polymyxin-agarose slurry in phosphate-buffered saline (PBS) was mixed with 1 ml of the purified SP-A preparation and incubated for 60 min at room temperature. The polymyxin-agarose was removed by centrifugation. The LPS content in the SP-A preparations was monitored before and after polymyxin treatment by the Limulus lysate assay (Associates of Cape Cod, Falmouth, MA) and contained <0.5 endotoxin unit/ml.

Preparation of rat RBMM. RBMM were isolated from female Sprague-Dawley rats as previously described (42). After differentiation in culture for 5 days, the RBMM were plated into 24-multiwell dishes at 5 × 105 cells/well.

Incubation of rat RBMM with mycobacteria. Frozen stocks of BCG or M. tuberculosis H37Ra were thawed and vortexed vigorously with a glass bead to break up any clumps. The mycobacteria were collected by centrifugation and then resuspended in DMEM. SP-A or buffer was added, and the mixture was incubated for 30 min at 37°C. Ingestion assays using H37Ra were performed as previously described for BCG in the presence and absence of SP-A (42). For BCG killing assays, SP-A-BCG complexes or BCG alone was added to RBMM at a ratio of 1:1 and incubated for 4 h in serum- and antibiotic-free DMEM. The cell monolayers were washed three times to remove noningested BCG, and fresh DMEM containing 10% fetal calf serum (without antibiotics) was added. For TNF-alpha and nitric oxide assays, mycobacteria or SP-A-mycobacteria complexes were incubated with RBMM for 24 or 48 h in serum- and antibiotic-free DMEM. Supernatants were removed and assayed for TNF-alpha by ELISA and for nitric oxide (as nitrite) using the Griess reagent as follows: 100 µl of medium were added to wells in a 96-well microtiter plate. Freshly prepared Griess reagent (1% sulfanilamide, 0.1% naphthylethylenediamide dihydrochloride, and 2.5% H3PO4; 100 µl) was added to the medium and incubated for 5-10 min at room temperature. The resulting absorbance was read at 540 nm. Nitrite concentrations were determined from a standard curve using sodium nitrite.

Mycobacterial killing assays. Two separate assays were used to assess killing of ingested BCG: 1) colony counts of isolated intracellular BCG and 2) uptake of [3H]uracil by intramacrophage BCG. For colony counts, cells containing BCG or SP-A-BCG were lysed by incubation in 400 µl of 0.25% SDS in 1.1 ml of Middlebrook medium for 10 min. BSA solution (500 µl of 40% BSA in PBS) was then added, and dilutions of the lysate were plated onto Middlebrook agar plates. Resulting colonies were enumerated after growth at 37°C for 2-3 wk. Because assessment of killing using colony counts is cumbersome and lengthy for determination of a large number of samples, a modification of the method of Chan et al. (5), using metabolic labeling of viable BCG, was used in assays with nitric oxide inhibitors as follows: cells were incubated with BCG or SP-A-BCG for 4 h at 37°C. The cells were washed, and DMEM containing 10% serum plus 2.5 µCi of [3H]uracil was added to each well. Assays were performed in quadruplicate. At various times from 1 to 5 days, the macrophage monolayers were dissolved in 0.25% SDS and the labeled BCG was collected on GF/C filters, washed extensively with water, dried, and counted in a liquid scintillation counter. In the original method of Chan et al. (5), significantly higher levels of labeled uracil incorporation were obtained due to higher ratios of BCG to macrophage (10:1). However, in our experiments these levels of BCG led to macrophage death over the time course being examined (up to 5 days). In addition, all of our previous ingestion and cytokine data were obtained using a BCG-to-macrophage ratio of 1:1. This resulted in low but highly reproducible levels of uracil incorporation and an effective way to assess BCG viability in these studies.

Western blot analysis. Cells were incubated with PBS, SP-A, BCG, or SP-A-BCG complexes for 24 h in serum- and antibiotic-free medium at a ratio of 1:1 BCG to macrophage and 20 µg of SP-A per 5 × 105 BCG. The cells were washed and then lysed in immunoprecipitation buffer (20 mM Tris, pH 7.75, containing 1% Triton X-100, 0.5% deoxycholate, 0.15 M NaCl, 0.02% sodium azide, and 0.34 trypsin inhibitory units aprotinin/ml). Protein concentration in the cell lysate was measured using the Bio-Rad dye method (Bio-Rad Laboratories, Richmond, CA), and equal amounts of protein were loaded per lane on a 4-20% SDS-acrylamide gel. Proteins were electrophoretically separated and then transferred to nitrocellulose. The nitrocellulose was blocked overnight and then probed with anti-iNOS antibody. The blot was washed and then incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG. Reactive bands were localized by chemiluminescence. Band densities were quantified using a Bio-Rad model 620 video densitometer.


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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

SP-A enhances BCG-induced production of TNF-alpha and nitric oxide. Several reports have demonstrated that TNF-alpha and nitric oxide are released by mycobacteria-infected rodent macrophages and that these mediators are involved in containment of mycobacterial growth (1, 5, 13). We examined the effect of SP-A on the release of these two mediators. Rat macrophages were incubated with BCG for 24 h in the presence of increasing doses of SP-A. As shown in Fig. 1, both TNF-alpha and nitric oxide levels in the medium were increased after treatment with SP-A-BCG complexes at every dose of SP-A compared with production by BCG alone. No effect on release of either mediator was found with SP-A alone (data not shown). We also investigated the effect of SP-A on both ingestion and mediator production by the avirulent H37Ra form of M. tuberculosis. As shown in Table 1, SP-A increased the ingestion of this pathogen and enhanced the production of TNF-alpha and nitric oxide after 24 h of infection.


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Fig. 1.   Surfactant-associated protein A (SP-A) enhances the production of inflammatory mediators induced by bacillus Calmette-Guerin (BCG). Rat bone marrow macrophages (RBMM; 5 × 105) in 24-well plates were incubated with BCG (1:1) or BCG preincubated with increasing concentrations of SP-A. Cells plus mycobacteria were incubated for 24 h in serum-free DMEM. Medium was removed at 24 h, and tumor necrosis factor-alpha (TNF) was measured by ELISA. Nitric oxide was measured using the Griess reagent. Results are the averages ± SD for 3 determinations and are representative of 4 separate experiments. , Nitric oxide; , TNF-alpha .


                              
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Table 1.   SP-A enhances ingestion of Mycobacterium tuberculosis H37Ra and production of NO and TNF-alpha

Anti-SPR210 antibodies inhibit SP-A-BCG-induced production of TNF-alpha and nitric oxide. We have reported previously the isolation and characterization of a receptor expressed on macrophages that mediates uptake of SP-A-BCG complexes (6, 42). To determine whether this receptor (SPR210) mediates the production of TNF-alpha and nitric oxide induced by SP-A-BCG complexes, rat macrophages were incubated with BCG alone or SP-A-BCG (20 µg of SP-A and 5 × 105 BCG) for 48 h in the presence of anti-SPR210 antibodies. As shown in Figs. 2 and 3, the SPR210 antibody blocked nitric oxide and TNF-alpha release induced in the presence of SP-A-BCG complexes. Normal rabbit serum had no effect on release of either mediator, and anti-SPR210 antibodies did not block LPS-induced production of nitric oxide (data not shown). In addition, the inhibitors L-NMMA and neutralizing anti-TNF-alpha antibodies blocked production of nitric oxide and TNF-alpha , respectively.


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Fig. 2.   Anti-SPR210 antibodies and N-monomethyl-L-arginine (L-NMMA) block the SP-A enhancement of BCG-induced nitric oxide production. RBMM (5 × 105) in 24-well plates were treated with medium alone, L-NMMA (250 µM), anti-SPR210 antibody (1:100), or normal rabbit serum (NRS; 1:100) followed by incubation with BCG (1:1) or BCG preincubated with 20 µg of SP-A for 48 h. Nitric oxide released into the medium was measured using the Griess reagent. NRS had no effect on nitric oxide production (data not shown). Results are the averages ± SD of 3 determinations and are representative of 3 separate experiments. * P < 0.001 for BCG-SP-A compared with BCG alone, BCG-SP-A (B/S) plus L-NMMA, and BCG-SP-A plus anti-SPR210.



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Fig. 3.   Anti-SPR210 antibodies and anti-TNF-alpha neutralizing antibodies block the SP-A enhancement of BCG-induced TNF-alpha production. RBMM were incubated as described in Fig. 2 with BCG alone, BCG-SP-A, BCG-SP-A with 1:500 anti-TNF-alpha antibodies, or BCG-SP-A with 1:100 anti-SPR210 antibodies. Results are the averages ± SD of 3 determinations and are representative of 3 separate experiments. * P < 0.001 for BCG-SP-A compared with BCG alone, BCG-SP-A with anti-TNF-alpha , or BCG-SP-A with anti-SPR210.

SP-A enhances the BCG-induced increase in iNOS protein expression. Nitric oxide production is dependent on the induction of synthesis of the iNOS enzyme (28). We examined the effect of SP-A, SP-A-BCG, and BCG alone on the levels of iNOS protein in rat macrophages. Macrophages (4 × 106 cells/well in 6-well dishes) were incubated for 24 h with BCG (1:1), SP-A-BCG (160 µg/4 × 106 BCG), PBS, or SP-A (160 µg). Nitrite levels were measured in the medium at 24 h, and the cells were solubilized in immunoprecipitation buffer for subsequent Western blotting with iNOS antibodies. As shown in Fig. 4, incubation of cells with SP-A or PBS did not stimulate iNOS synthesis (lanes 1 and 2). iNOS protein appeared following stimulation with BCG and was increased ~1.5-fold by SP-A addition to the BCG. Nitrite levels were increased 1.8-fold in the same experiment (data not shown). These results indicate that SP-A enhances BCG-induced production of nitric oxide through induction of iNOS protein and that SP-A alone has no effect on either nitric oxide production or induction of iNOS protein.


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Fig. 4.   SP-A enhances the BCG-induced increase in inducible nitric oxide synthase (iNOS). RBMM (4 × 106) were incubated with BCG (1:1), BCG preincubated with SP-A (160 µg/4 × 106 BCG), SP-A (160 µg), or PBS in serum-free and antibiotic-free medium. Medium was removed at 24 h for nitrite determination, and cells were lysed in immunoprecipitation buffer (100 µl). An aliquot of each cell lysate was added to an equal volume of sample buffer and then electrophoresed on a 4-20% gradient polyacrylamide reducing gel. Proteins were transferred to nitrocellulose and probed with anti-iNOS antibodies at a 1:10,000 dilution. After incubation with secondary antibody, blots were developed by chemiluminescence. Equal amounts of cellular protein were added to each lane. Relative density of each band was determined by densitometric scanning. The experiment shown is representative of 2 separate experiments. Lane 1, PBS control; lane 2, SP-A alone; lane 3, BCG alone; lane 4, SP-A-BCG.

SP-A enhances the killing of internalized BCG. In our previous studies, we demonstrated that opsonization of BCG with SP-A enhanced ingestion by rat macrophages (42). To investigate the fate of the internalized BCG or SP-A-BCG complexes, we assessed the growth rate of the intramacrophage BCG by colony counts. Macrophages were incubated with SP-A-BCG complexes or BCG alone for 4 h, and the cultures were washed and then fed with antibiotic-free medium. On days 1-5, the macrophages were lysed with SDS and the released mycobacteria were plated onto Middlebrook agar plates. Colonies were enumerated after 2-3 wk of growth. As shown in Fig. 5, in the absence of SP-A, intracellular BCG showed increased growth over the 5-day period. In the presence of SP-A, the initial level of intramacrophage BCG was approximately fourfold higher than BCG alone, reflecting the enhanced ingestion with SP-A. However, by day 5, the number of BCG had fallen to one-half the number at day 1. The number of macrophages per well was not significantly different between the BCG and SP-A-BCG groups, indicating that the increase in BCG ingestion had not led to macrophage killing. These results indicate that the presence of SP-A during the initial ingestion phase led to an enhanced rate of killing of the internalized BCG.


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Fig. 5.   SP-A enhances the killing of ingested BCG. RBMM were incubated with BCG (1:1) or BCG preincubated with SP-A (20 µg) for 4 h. The unbound BCG were removed by washing, and the cultures were incubated in fresh medium with serum and without antibiotics for an additional 1-5 days. At days 1, 2, and 5, the macrophages were lysed with SDS, and the released viable BCG were plated onto Middlebrook agar plates. Colonies were enumerated after growth for 2-3 wk. Results are the averages ± SD of 3 determinations and are representative of 2 separate experiments.

Inhibition of nitric oxide production blocks BCG killing. A number of laboratories have reported that murine macrophages kill mycobacteria through a nitric oxide-dependent mechanism (1, 5, 13). In the results presented above, we have shown that BCG infection of RBMM leads to production of nitric oxide and that levels of this mediator are increased when the BCG organisms are complexed with SP-A. We therefore examined the role of nitric oxide in the SP-A-enhanced killing of BCG. Cells were incubated with BCG alone or SP-A-BCG for 4 h, and then intramacrophage BCG was labeled up to 5 days by incubation with [3H]uracil. Uracil incorporation by BCG that had been ingested in the presence of SP-A was higher at days 3 and 4, reflecting the SP-A-enhanced ingestion. However, at day 5, uracil incorporation by viable BCG from SP-A-BCG cultures had fallen below the levels for BCG alone as shown in Fig. 6, supporting the previous data demonstrating increased killing by SP-A (Fig. 5). When cultures were incubated with the iNOS inhibitor L-NMMA, numbers of viable BCG from macrophages incubated with either BCG alone (data not shown) or SP-A-BCG complexes were significantly higher, demonstrating that inhibition of nitric oxide production blocked killing of BCG by rat macrophages.


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Fig. 6.   L-NMMA blocks BCG killing in the presence of SP-A. RBMM were incubated with BCG or SP-A-BCG (B/S) complexes as described in Fig. 4. After removal of unbound BCG, cells plus ingested organisms were refed with fresh medium minus antibiotics plus serum containing 2 µCi/well of [3H]uracil. L-NMMA (250 µM) was added to one-half of the BCG and SP-A-BCG wells. At days 3, 4, and 5, macrophages were lysed with SDS, and viable BCG were collected by filtration over GF/C filters. The filters were dried and then counted by liquid scintillation counting. Nitric oxide production was monitored on days 1-5 throughout the experiment, and viability of macrophages in companion wells was verified by vital dye exclusion. Results shown for day 5 are the averages ± SD of 4 determinations and are representative of 2 separate experiments. * P < 0.001 for BCG compared with SP-A-BCG; ** P < 0.001 for SP-A-BCG plus L-NMMA compared with BCG and SP-A-BCG.


    DISCUSSION
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ABSTRACT
INTRODUCTION
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Mycobacteria use macrophage cell surface receptors to gain entry into the cell where they either survive and replicate in the phagosomal compartment or are killed by macrophage-derived products. The receptors used to gain entry may determine the ultimate fate of the internalized organisms. In this report, we have demonstrated that BCG ingestion by rat macrophages results in the production of the inflammatory mediators TNF-alpha and nitric oxide. Nitric oxide appears to be involved in killing of the internalized BCG because the NOS inhibitor L-NMMA blocks subsequent mycobacterial killing. Opsonization of the BCG with SP-A dramatically increases levels of both TNF-alpha and nitric oxide and enhances BCG killing. These effects may be mediated in part by the recently described receptor for SP-A, SPR210, because antibodies against the receptor block the SP-A-enhanced ingestion of BCG (42) and subsequent release of TNF-alpha and nitric oxide.

In addition to the current study of BCG ingestion, several recent reports have shown that SP-A enhances killing of pathogens by macrophages. Hickman-Davis et al. (16) reported that SP-A binding to IFN-gamma -treated murine alveolar macrophages increased killing of ingested mycoplasma through a nitric oxide-dependent pathway. Kabha et al. (17) showed that SP-A enhanced the phagocytosis and killing of K. pneumoniae through a direct interaction of SP-A-K. pneumoniae complexes with the macrophages as well as the phagocytosis by preincubation of cells with SP-A. The mechanisms involved in Klebsiella killing were not investigated, but previous studies have shown that nitric oxide is involved (38). In recent in vivo studies using SP-A-deficient mice, LeVine and co-workers (20-22) have reported that clearance of three pulmonary pathogens, RSV, P. aeruginosa, and group B streptococcus (GBS), is dramatically impaired in the absence of SP-A. Furthermore, the number of alveolar macrophages with phagocytosed GBS was lower in SP-A(-/-) mice, and opsonization of GBS with SP-A increased the phagocytosis by SP-A(-/-) alveolar macrophages (23). All of these studies support the current results, demonstrating a direct beneficial effect of SP-A for the host through enhanced pathogen killing.

We have shown that both BCG and SP-A-BCG complexes trigger production of nitric oxide, which appears to be involved in the killing and inhibition of growth of ingested BCG organisms. Reactive nitrogen intermediates (RNI) have been implicated in the antimicrobial effect of activated macrophages against a wide variety of infections in mice (8, 28), and involvement of nitric oxide in containment of mycobacterial infections has been demonstrated by several groups. Activated murine macrophages inhibited the growth of and killed intracellular mycobacteria organisms, and with use of L-NMMA as an inhibitor of iNOS, RNI were shown to be the principal effector mechanism responsible for this antimicrobial activity (1, 5, 9, 13). A role for RNI in vivo was suggested by the recent report by MacMicking et al. (24) who demonstrated that mice lacking the iNOS gene were highly susceptible to M. tuberculosis infection. The results in the present study not only support a role for macrophage-derived nitric oxide in mycobacterial killing, but more importantly, they demonstrate the involvement of SP-A in initiation of signaling for nitric oxide production. A recent study by Pasula et al. (31) has also addressed the role of SP-A in lung host defense against mycobacteria. In this study, SP-A suppressed nitric oxide by IFN-gamma -activated mouse alveolar macrophages in response to M. tuberculosis. In the current study, we have examined the effect of SP-A on interaction of mycobacteria with nonactivated macrophages. It is possible that prior activation by endogenously produced cytokines may alter the response of the infected macrophage to SP-A. We have previously shown that IFN-gamma increases SP-A binding to macrophages (7), potentially altering the signaling mechanisms or routes of entry for SP-A-mycobacterial complexes.

The mechanisms involved in SP-A-mediated signaling, resulting in production of nitric oxide, are not known. We have preliminary evidence suggesting that tyrosine kinase pathways might be activated, supporting the recent report by Schagat et al. (36) that tyrosine kinase inhibitors block SP-A-stimulated phagocytosis and SP-A-directed chemotaxis in alveolar macrophages. Activation of tyrosine kinases and subsequent cellular protein phosphorylation appear to be common pathways in signal transduction initiated by ligand binding to a variety of phagocytic receptors. For example, LPS activation of murine macrophages induces tyrosine-directed protein phosphorylation, and inhibition of this phosphorylation blocks nitric oxide production (10).

The mechanism of SP-A-mediated enhancement of phagocytosis has not yet been clearly defined. Results from our studies and others support an opsonin role for SP-A (17, 21, 27, 32, 37, 39), whereas other groups have suggested that SP-A first binds to a cell surface receptor followed by increased expression of other receptors that mediate internalization of unopsonized pathogens (14, 17, 40). Although we are suggesting that SP-A enhances mycobacterial ingestion and killing via an opsonin-like process, it is possible that SP-A functions as both an opsonin and a stimulator of phagocytosis through distinct mechanisms. First, BCG might be interacting with its own receptor, which causes the appearance of SP-A-specific signaling receptors. This is supported by the recent report by Plaga et al. (33) that at least two separate receptors with differing SP-A binding properties can be isolated from bovine macrophages. One receptor might be involved in clearance of extracellular SP-A or SP-A-particulate complexes, with no accompanying cytokine production. A second receptor could be specifically involved in SP-A-mediated signaling events. However, our findings that anti-SPR210 blocks soluble SP-A binding, SP-A-BCG ingestion, and SP-A-BCG-induced mediator production would suggest that a single receptor, SPR210, is mediating the SP-A-mycobacterial effects. Second, SP-A binding to SPR210 or another receptor might enhance or upregulate a BCG-specific receptor on the macrophage surface. For example, Gaynor et al. (14) reported that SP-A pretreatment of macrophages led to a rapid increase in cell surface mannose receptors that participated in phagocytosis of M. tuberculosis, and in the study by Kabha et al. (17), SP-A acted not only as an opsonin for K. pneumoniae but also appeared to increase surface expression of mannose receptors.

In the current study, we observed that BCG in the absence of any added opsonins was ingested by macrophages, and this uptake was accompanied by the production of nitric oxide and inhibition of growth as evidenced by the increase in viable organisms in the presence of L-NMMA (data not shown). The identity of this receptor is not known. Prinzis et al. (34) have reported that the structure of the lipoarabinomannan in BCG contains the same mannose capping found in M. tuberculosis, suggesting that this would present a potential ligand for the macrophage mannose receptor. In support of the involvement of the mannose receptor, Venisse et al. (41) have recently reported that BCG interacts with a Ca2+-dependent recycling receptor in a mannose-dependent manner. Studies are currently underway to determine the involvement of this receptor in BCG uptake and signaling.

In the present study, we found no effect of SP-A alone on mediator production by RBMM, although SP-A-mycobacteria complexes did induce production of nitric oxide and TNF-alpha . Signaling initiated by binding of particulate ligands but not of soluble ligands has been reported for other receptors, including the mannose receptor (12) and the Fcgamma RIII receptor (35). It has been postulated that particulate ligands may induce receptor cross-linking and a subsequent association with intracellular signaling pathways that is not induced by soluble ligand binding. Other studies have, however, reported responses of macrophages treated with SP-A alone. This is a controversial area, perhaps due to at least two major factors: 1) the preparation and source of SP-A and 2) the source of macrophages used for the study. In most of our experiments, we have used rat marrow macrophages, but in every case we have demonstrated that the effects are also seen in rat alveolar macrophages. We have used SP-A prepared from human proteinosis fluid using a butanol extraction procedure. Our finding that SP-A has no effect on mediator production is in agreement with studies by McIntosh et al. (25) who found no stimulation of TNF-alpha production after treatment of rat macrophages with human SP-A. Similarly, Weissbach et al. (43) found no effect of purified rat or dog SP-A on superoxide production by rat alveolar macrophages. In contrast, Koptides et al. (18) and Kremlev and Phelps (19) reported that cytokine production and NF-kappa B activation were enhanced when human monocytes or human THP-1 monocyte-like cells were exposed to human SP-A prepared by preparative isoelectric focusing. In addition, Blau et al. (3) reported that both human and rat SP-A increased nitric oxide production by rat alveolar macrophages exposed to rat or human SP-A. In all cases, LPS contamination was ruled out by careful monitoring of LPS levels. It is apparent that a combination of factors is contributing to the discrepancies in these data, and interpretation of results must take into consideration the experimental conditions used.

A number of laboratories have demonstrated that SP-A has the ability to alter macrophage functions and to act as an opsonin to enhance ingestion of various pulmonary pathogens. The finding that mice carrying a disrupted SP-A gene cannot clear GBS, RSV, or P. aeruginosa from their lungs underscores the potential role of this lectin in pulmonary host defense (20-23). Recent evidence that other lung lectins such as soluble SP-D (15) and the cell surface mannose receptor (12) can bind pathogens and promote their ingestion suggests that multiple mechanisms exist in the lung to recognize invading microbes. For example, SP-A levels are decreased during pulmonary infections and other disease states (44), and expression of both the mannose receptor and the SPR210 are tightly linked to the functional state of the macrophage (7). It is probable, therefore, that pulmonary host defense is dependent on a variety of factors, including availability of cell surface and soluble lectins for interactions with pathogens, local cytokine levels that will control microbicidal activity of the macrophage, and availability of phagocytic receptors that will lead to intracellular killing of invading pathogens.


    ACKNOWLEDGEMENTS

This work was supported in part by National Heart, Lung, and Blood Institute Grant HL-55977 and a Merit Review Award from the Department of Veterans Affairs (V. L. Shepherd).


    FOOTNOTES

Address for reprint requests and other correspondence: V. L. Shepherd, VA Medical Center/Research Service, 1310 24th Ave S, Nashville, TN 37212 (E-mail: virginia.l.shepherd{at}vanderbilt.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Received 12 July 1999; accepted in final form 21 February 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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