Nitrated SP-A does not enhance adherence of Pneumocystis carinii to alveolar macrophages

Departments of ¹ Anesthesiology, ³ Physiology and Biophysics, and ⁴ Pediatrics, University of Alabama at Birmingham, Birmingham, Alabama 35233; and ² Division of Pulmonary, Critical Care, and Occupational Medicine, Department of Medicine, Indiana University School of Medicine, Indianapolis, Indiana 46202

	ABSTRACT

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We investigated whether nitration of surfactant apoprotein (SP) A alters its ability to bind to mannose-containing saccharides on Pneumocystis carinii and its potential role in the mediation of P. carinii adherence to alveolar macrophages. Human SP-A was nitrated by incubation with tetranitromethane at pH 8.0 or synthetic peroxynitrite (ONOO) at pH 7.4, which resulted in significant nitration of tyrosines in its carbohydrate recognition domain [0.63 ± 0.001 (SE) and 1.25 ± 0.02 mol nitrotyrosine/mol monomeric SP-A, respectively; n = 3 samples]. Binding of SP-A to P. carinii was calcium dependent and competitively inhibited by -methyl-D-mannopyranoside. Nitration of SP-A by ONOO or tetranitromethane decreases its binding to P. carinii by increasing its dissociation constant from 7.8 × 10⁹ to 1.6 × 10⁸ or 2.4 × 10⁸ M, respectively, without significantly affecting the number of binding sites (7.1 × 10⁶/P. carinii organisms, assuming that the native molecular mass of oligomeric SP-A is 650 kDa). Furthermore, ONOO-nitrated SP-A failed to mediate the adherence and phagocytosis of P. carinii to rat alveolar macrophages as observed with normal SP-A. Binding of SP-A to rat alveolar macrophages was not altered by nitration. These results indicate that nitration of SP-A interferes with its ability to serve as a ligand for P. carinii adherence to alveolar macrophages at the site of the SP-A moleculeP. carinii interaction.

surfactant protein A; collectin; tyrosine nitration; parasite adherence; nitric oxide; lung host defense

	INTRODUCTION

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pneumocystis carinii PNEUMONIA remains a common and life-threatening pulmonary infection in immunocompromised patients, especially those with acquired immunodeficiency syndrome. The interaction between P. carinii and alveolar macrophages within the alveolar lining fluid of the lung represents the initial contact of the pathogen with the host immune system.

Alveolar macrophages express many different types of surface receptors that aid in their ability to bind to microorganisms. Among these are receptors for mannose (50), fibronectin (4), the Fc portion of IgG (38), the complement component C3b (66), and surfactant protein (SP) A (5). Alveolar macrophages have been shown to play a significant role in host defense by binding, phagocytosing, and degrading P. carinii (30, 58, 63).

The predominant surface membrane protein on P. carinii is a glycoprotein with an estimated molecular mass of 110-120 kDa known as major surface glycoprotein (MSG) (46). A recent study (42) suggested that MSG may serve to mediate adherence of P. carinii to the alveolar epithelium. P. carinii MSG is heavily glycosylated with mannose-containing oligosaccharide chains (46) that could function as a ligand for SP-A (69).

SP-A, the most abundant apoprotein of the pulmonary surfactants, is a member of the C-type lectin superfamily (7, 63) and along with SP-D (40), serum mannose-binding protein (MBP) A, MBP-C, conglutinin, and the recently described collectin-43 (22) forms the collectin (group III) subgroup (7, 31). The human SP-A molecule is organized into four discrete structural domains: a short amino-terminal globular domain containing a single cysteine involved in interchain disulfide bond formation, a collagen-like domain, a hydrophobic neck region, and a carboxy-terminal carbohydrate recognition domain (CRD). Thus SP-A is a lectin protein with a collagen-like domain that shares extensive structural homology with MBPs (8), conglutinin (6), and complement component C1q (56). It has multiple functions including tubular myelin formation, binding to high-affinity receptors on alveolar type II cells, regulating the recycling of surfactant lipids, and acting synergistically with other surfactant apoproteins to lower surface tension (15, 20, 28, 64). Recent studies have shown that SP-A is implicated in lung host defense by interacting with a variety of pathogens (34, 35, 54, 63, 69) and stimulating chemotaxis, phagocytosis, and production of reactive oxygen species by alveolar macrophages (32, 34, 52, 53, 59, 63, 65).

Exposure of alveolar macrophages and airway and alveolar cells to diverse stimuli of inflammation such as cytokines (interleukin-1, tumor necrosis factor-, and interferon-) and lipopolysaccharide (LPS) results in a marked upregulation of nitric oxide (· NO) and superoxide (O₂·) production (1, 14, 45, 48, 57, 63). The product of the reaction of · NO with O₂·, which proceeds at a near diffusion-limited rate (6.7 × 10⁹ M/s) (39), is peroxynitrite (ONOO), a potent oxidizing and nitrating agent that damages a wide spectrum of biological molecules such as DNA (23), lipids (47), and proteins (12, 36). Recent studies (16, 17, 37, 67) have indicated that exposure of SP-A to nitrating reagents such as ONOO, tetranitromethane (TNM), or nitrogen dioxide (· NO₂) results in a marked decrease in its ability to aggregate lipids and bind mannose. Thus we hypothesized that nitration of SP-A would impair its ability to bind to P. carinii, a step necessary for the clearance of these organisms by alveolar macrophages.

In the present study we have 1) characterized the binding of normal and nitrated SP-A to both P. carinii and alveolar macrophages, 2) determined the effect of SP-A on the interaction of P. carinii with alveolar macrophages, and 3) assessed which structural domain of SP-A is involved in these processes. We found that human SP-A can bind to P. carinii through its CRD and that nitration of tyrosine residues in the CRD decreases its affinity for mannose-rich structures on the surface of P. carinii. Furthermore, normal SP-A enhances the adherence and phagocytosis of P. carinii to alveolar macrophages, whereas nitrated SP-A fails to mediate this process. Because the binding of nitrated SP-A to alveolar macrophages is not significantly affected, it is likely that nitration of SP-A interferes with its ability to serve as a ligand for P. carinii adherence to alveolar macrophages at the site of the SP-A molecule-P. carinii interaction.

	METHODS

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Materials. BSA, HEPES, EDTA, EGTA, DMEM, bisbenzimide (Hoechst 33258), -methyl-D-mannopyranoside, o-phenylenediamine, and LPS (serotype 055:B5 from Escherichia coli) were from Sigma (St. Louis, MO). Sodium nitrite and hydrogen peroxide were obtained from Fisher Scientific (Fair Lawn, NJ). TNM and 3-nitro-L-tyrosine were from Aldrich (Milwaukee, WI). Nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl-1-phosphate kit came from Promega (Madison, WI). Hanks' balanced salt solution (HBSS) without calcium or magnesium was from GIBCO BRL (Grand Island, NY). Bicinchoninic acid protein assay kit was from Pierce Chemical (Rockford, IL). ⁵¹Cr-labeled sodium chromate was from NEN (Boston, MA). Goat anti-rabbit IgG and Tween 20 were from Bio-Rad (Richmond, CA). Immulon 2 ELISA plates were from Dynatech (Chantilly, VA). Rabbit anti-human SP-A and anti-nitrotyrosine were kind gifts of Drs. D. S. Phelps (Pennsylvania State University, Hershey, PA) and J. S. Beckman (University of Alabama at Birmingham, Birmingham, AL), respectively.

Isolation of P. carinii. P. carinii pneumonia was induced in Lewis rats (Harlan Sprague Dawley, Indianapolis, IN) by immunosuppression with dexamethasone and transtracheal inoculation of P. carinii. P. carinii were isolated, purified as previously described (60), and washed extensively with HBSS containing 5 mM EGTA to remove endogenously bound rat SP-A (33). Purification of the P. carinii suspension results in >98% of the cellular material representing trophozoites (43, 44). P. carinii were quantified by the method of Bartlett et al. (2). A typical yield was 2 × 10⁷ P. carinii organisms/rat. Any preparations found to contain bacterial, fungal, or inflammatory cell contamination were discarded.

Purification of human SP-A. SP-A was purified from bronchoalveolar lavage fluid of patients with alveolar proteinosis by n-butanol extraction as previously described (17). SP-A was dissolved in 10 mM HEPES buffer, pH 7.4, the protein concentration was determined by the bicinchoninic acid method, and the samples were stored in small aliquots at 20°C until used. The purity of SP-A was demonstrated by SDS-PAGE and Western blotting, and the function was checked by its lipid aggregation and mannose-binding abilities (16, 19, 67). the endotoxin level tested negative (<0.01 endotoxin unit/ml) with the amebocyte lysate assay performed by the University of Alabama at Birmingham Media Preparation Shared Facility.

Synthesis of ONOO. ONOO was synthesized from sodium nitrite and hydrogen peroxide with a quenched-flow reactor as previously described (3) and treated with manganese dioxide to remove contaminated hydrogen peroxide. The ONOO concentration was determined spectrophotometrically at 302 nm (molar extinction coefficient = 1,670 M/cm) before each experiment.

Exposure of SP-A to nitrating agents. SP-A (1 mg/ml) in 10 mM HEPES buffer was incubated with TNM or ONOO at 37°C in the following fashion: 1) 0.5 mM TNM at pH 8.0 for 30 min or 0.5% ethanol as a vehicle control or 2) 0.5 mM active or inactive ONOO at pH 7.4 for 15 min. ONOO was inactivated by diluting the stock solution 20-fold in 10 mM HEPES buffer, pH 7.4, and heating the solution to 37°C for 20 min. Absorbance measurements verified that >99% of ONOO was inactivated. A second dose of active or inactive ONOO (0.5 mM) was added into the SP-A-containing solutions at the end of the 15-min period, and the solutions remained at 37°C for an additional 15 min. SP-A protein was either used immediately after exposure or stored at 20°C for later use.

Binding of SP-A to P. carinii. For binding experiments, 5 × 10⁴ freshly isolated P. carinii organisms and 100-1,000 ng of SP-A were mixed in 100 µl of either binding (HBSS without calcium or magnesium containing 1% BSA and 5 mM CaCl₂, pH 7.4) or EGTA buffer (HBSS without calcium or magnesium, containing 1% BSA and 5 mM EGTA, pH 7.4). The sedimentation of SP-A due to its self-association was also measured in the presence of 5 mM CaCl₂ but in the absence of P. carinii. Total binding (calcium-dependent and -independent binding) was always corrected for by the sedimentation of SP-A.

Because our preliminary experiments showed that human SP-A binds to P. carinii in a time-dependent fashion and that binding plateaued in 30 min, the reaction mixtures were incubated for 30 min in an atmosphere of 95% air-5% CO₂ at 37°C. After incubation, the reaction mixtures were centrifuged at 13,000 g for 5 min. The pellets were washed three more times with either binding or EGTA buffer. The pellets were then resuspended in water and sonicated briefly to homogenize P. carinii. SP-A was determined by a capture enzyme-linked immunosorbent assay (ELISA) as described in Quantification of SP-A and SP-A nitration. Specific binding was defined as the calcium-dependent binding and calculated by subtracting the calcium-independent binding (binding in the presence of 5 mM EGTA) from the total binding (which was already corrected for nonspecific SP-A sedimentation). Calcium-independent binding plus nonspecific sedimentation of SP-A was found to be <25% of the binding in the presence of 5 mM CaCl₂. Binding assays also were performed in the presence of 0-750 mM -methyl-D-mannopyranoside in 100 µl of binding buffer containing 1 × 10⁶ P. carinii organisms and 100 ng of SP-A.

Quantification of SP-A and SP-A nitration. The amount of SP-A was determined by ELISA with a polyclonal rabbit anti-human SP-A as the primary antibody and horseradish peroxidase-conjugated goat anti-rabbit IgG as the secondary antibody. In brief, samples were serially diluted and coated to ELISA plates and allowed to bind for at least 18 h at 4°C. Nonspecific binding sites were blocked with 1% BSA for 1 h at room temperature. The wells were then incubated with the primary antibody (1:5,000 dilution) at 37°C for 1 h, followed by the secondary antibody (1:2,500 dilution). Hydrogen peroxide and o-phenylenediamine were used as substrates for the peroxidase reaction, and the absorbance was measured at 490 nm. Purified SP-A was used as the standard. Additional experiments also showed that the polyclonal rabbit anti-human SP-A antibody we used for SP-A quantification had similar affinities for both normal and nitrated SP-A (data not shown).

Nitrotyrosine content of TNM- or ONOO-treated SP-A was measured by ELISA with the nitrotyrosine antibody instead of the SP-A antibody as the primary antibody and horseradish peroxidase-conjugated goat anti-rabbit IgG as the secondary antibody as previously described (16, 67).

Isolation of alveolar macrophages. Lungs from Lewis rats (Harlan Sprague Dawley) were lavaged, and the alveolar cells were concentrated by centrifugation. The cells were washed three more times with HBSS containing 5 mM EGTA to remove endogenously bound rat SP-A. Examination of cytopreparation smears stained with Diff-Quik demonstrated that >95% of the cells obtained were macrophages. These cells were then plated at a density of 1 × 10⁶ cells/well in DMEM supplemented with 25 mM NaHCO₃, 15 mM HEPES, 100 U/ml of penicillin, 100 µg/ml of streptomycin, 4 µg/ml of gentamicin, and 0.5 µg/ml of amphotericin B, pH 7.4, on rat IgG-coated 24-well tissue culture plates and allowed to adhere for 1 h at 37°C. After adherence, each well was washed twice with the same medium to remove unattached cells, then used immediately for SP-A-binding experiments or cultured overnight in a P. carinii adherence and phagocytosis assay.

SP-A binding to alveolar macrophages. The culture plates were placed on ice and washed three times with cold binding medium (same DMEM medium containing 0.1% BSA). The cells were then incubated with varying concentrations (0-15 µg/ml) of SP-A in binding medium at 4°C for 1 h. At the end of the incubation, the cells were washed four times with HBSS containing 1.8 mM of calcium and reconstituted in water by brief sonication. SP-A bound to alveolar macrophages was quantified by ELISA as described in Quantification of SP-A and SP-A nitration. The total binding of SP-A to macrophages was normalized by the cell DNA content (see method in DNA measurement). Nonspecific binding of SP-A to the plate wells in the absence of cells was <10% of the total binding.

P. carinii adherence and phagocytosis to alveolar macrophages. P. carinii adherence to alveolar macrophages was assayed with ⁵¹Cr-labeled P. carinii. Freshly isolated P. carinii were incubated with ⁵¹Cr-labeled sodium chromate (50 µCi/ml) in 1 ml of DMEM with fetal bovine serum overnight. The radiolabeled P. carinii were then extensively washed with DMEM containing 5 mM EGTA to remove endogenous rat SP-A and other surface proteins along with any nonincorporated ⁵¹Cr as previously described (42, 60). Normal or ONOO-nitrated SP-A was added to each designated well followed by the immediate addition of ⁵¹Cr-labeled P. carinii (5 × 10⁶ organisms/well). The adherence and phagocytosis assay was conducted in DMEM containing 0.1% BSA but no fetal bovine serum. Because the adherence of P. carinii to rat alveolar macrophages plateaued in 4 h (60) and the phagocytosis reached plateau in 2 h, the plates were briefly centrifuged at 800 g for 5 min and incubated at 4°C for 4 h to determine adherence or at 37°C for 2 h to determine adherence/phagocytosis. After the incubation period, adhered macrophages were washed with normal saline three times. The supernatant and subsequent washes were pooled and labeled as fraction A. The cells were lysed with 10% Triton X-100 and labeled as fraction B. Radioactivity was quantified in a gamma counter (Beckman 5500, Beckman Instruments, Schaumburg, IL). The percentage of adherence or adherence and phagocytosis was calculated as follows: [fraction B/(fraction A + fraction B)] × 100. Each experiment was performed in duplicate and repeated on at least three separate occasions.

SDS-PAGE and Western blotting. EGTA-washed P. carinii or macrophages were solubilized in 50 mM Tris · HCl buffer, pH 6.8, containing 5% -mecaptoethanol, 2% SDS, 0.01% bromphenol blue, and 10% glycerol and were resolved by 12% SDS-PAGE as previously described (67). The gels were either stained with 0.25% Coomassie brilliant blue R-250 (for SDS-PAGE) or electrophoretically transferred onto nitrocellulose membranes (for Western blotting). Nonspecific protein binding sites on the nitrocellulose membranes were blocked by 5% dry milk in 10 mM Tris · HCl buffer containing 150 mM NaCl and 0.05% Tween 20, pH 8.0, for at least 2 h. The nitrocellulose-bound antigen was overlaid with a rabbit anti-human SP-A antibody (1:10,000 dilution) followed by alkaline phosphatase-conjugated goat anti-rabbit IgG (1:7,500 dilution) as the secondary antibody. As a control, the primary antibody was replaced by the same concentration of nonspecific rabbit IgG. Bound antibody was detected with nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl-1-phosphate kit.

DNA measurement. Cellular DNA was determined according to the method of Labarca and Paigen (29) by measuring the enhanced fluorescence of bisbenzimide (Hoechst 33258) bound to the DNA. The excitation and emission wavelengths used were 356 and 458 nm, respectively.

Statistical analysis. Significant differences among group means were determined by one-way analysis of variance and the Bonferroni modification of the t-test. Results are expressed as means ± SE. P < 0.05 was considered to be significantly different from control values.

	RESULTS

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EGTA removal of endogenously bound rat SP-A from P. carinii and alveolar macrophages. SDS-PAGE and Western blotting studies shown in Fig. 1 indicated that washing P. carinii and alveolar macrophages with HBSS containing EGTA removed all surface-associated SP-A. This is in agreement with previous results (60).

View larger version (71K): [in this window] [in a new window]   Fig. 1.   SDS-PAGE (A) and Western blotting (B) confirmation of EGTA removal of endogenously bound rat surfactant protein (SP) A from Pneumocystis carinii and rat alveolar macrophages. Isolated P. carinii and macrophages were washed extensively with Hanks' balanced salt solution (HBSS) containing 5 mM EGTA. SDS-PAGE and Western blotting were performed under reducing conditions as described in METHODS. SP-A was detected with a rabbit polyclonal anti-human SP-A antibody that cross-reacts with rat SP-A. Lane 1, EGTA-washed P. carinii (5 × 106 P. carinii organisms); lane 2, EGTA-washed alveolar macrophages (1 × 105 macrophages); lane 3, purified human SP-A (0.5 µg). Nos. at left, molecular-mass markers.

SP-A nitration. Unexposed SP-A or SP-A treated with inactive ONOO contained background levels of nitrotyrosine (<0.001 mol nitrotyrosine/mol monomeric SP-A). In contrast, SP-A exposed to two boluses of 0.5 mM ONOO or a single dose of 0.5 mM TNM contained significant levels of nitrotyrosine as measured by ELISA (0.63 ± 0.001 and 1.25 ± 0.02 mol nitrotyrosine/mol monomeric SP-A, respectively; n = 3 samples; Fig. 2A).

View larger version (25K): [in this window] [in a new window]   View larger version (13K): [in this window] [in a new window]   Fig. 2.   A: total (calcium dependent and independent) and calcium-independent binding of normal and nitrated (SP-ABound) SP-A to P. carinii. SP-A was nitrated by exposure to peroxynitrite (ONOO) or tetranitromethane (TNM) as described in METHODS. Binding assays were performed with 5 × 104 P. carinii organisms and 100 ng of SP-A in 100 µl of HBSS containing 1% BSA and 5 mM either CaCl2 or EGTA (calcium-independent binding). Total binding (calcium dependent and independent) was corrected for nonspecific sedimentation of SP-A in presence of 5 mM CaCl2 but in absence of P. carinii. Nos. above bars, mean values of SP-A nitrotyrosine content (in mol nitrotyrosine/mol monomeric SP-A) as measured by ELISA. Nitration of SP-A resulted in a decrease in total but not in calcium-independent binding. Results are means ± SE of 3 experiments performed in duplicate. ** Significant difference compared with corresponding group in presence of calcium, P < 0.01. Significant difference compared with normal SP-A in the presence of calcium: # P < 0.05; ## P < 0.01. B: correlation between SP-A nitration and inhibition of its specific binding (SP-ASpecific) to P. carinii. Results are means ± SE of 3 experiments performed in duplicate. Significant difference compared with normal control SP-A: * P < 0.05; ** P < 0.01.

SP-A binding to P. carinii. Figure 2A also shows the calcium dependence of SP-A binding to P. carinii. The total binding in the presence of 5 mM CaCl₂ was corrected for sedimentation of SP-A due to its self-association. Binding of control SP-A to P. carinii was decreased by 84% in the presence of 5 mM of EGTA (P < 0.01), indicating a calcium-dependent process and the surface nature of the binding (69). Nitration of SP-A by ONOO or TNM significantly decreased its total binding in the presence of 5 mM CaCl₂ but not its calcium-independent binding in the presence of 5 mM EGTA. Furthermore, the extent of decrease in the SP-A-specific (i.e., calcium-dependent) binding to P. carinii was correlated with SP-A nitrotyrosine levels (Fig. 2B).

To clarify a potential mechanism for the decreased binding of nitrated SP-A to P. carinii, Scatchard analysis was performed. As shown in Fig. 3A, both normal and nitrated SP-A bound to P. carinii in a specific and saturable fashion. Scatchard plots of specific-binding data (i.e., calcium-dependent binding) are linear, suggesting a homogeneous population of binding sites for both normal and nitrated SP-A on P. carinii (Fig. 3B). Normal SP-A bound P. carinii with a binding dissociation constant (K_d) of 7.8 × 10⁹ M. The estimated number of SP-A binding sites was 7.1 × 10⁶/P. carinii organisms, assuming that the native molecular mass of oligomeric SP-A is 650 kDa (25). Nitration of SP-A by ONOO or TNM decreased its binding to P. carinii by increasing the K_d value to 1.6 × 10⁸ and 2.4 × 10⁸ M, respectively, without significantly affecting the number of binding sites. The magnitude of the K_d increase correlated with nitrotyrosine levels in SP-A. These results indicate that modification of tyrosine residues by nitrating agents in the CRD of SP-A decreased its affinity for SP-A binding sites on P. carinii.

View larger version (20K): [in this window] [in a new window]   View larger version (20K): [in this window] [in a new window]   Fig. 3.   A: specific binding of normal, TNM-treated, or ONOO-treated SP-A to P. carinii. Binding assays were performed with 5 × 104 P. carinii and 100-1,000 ng of SP-A (SP-AAdded) in 100 µl of HBSS containing 1% BSA and 5 mM either CaCl2 or EGTA. Total binding (calcium dependent and independent) was always corrected for SP-A sedimentation in presence of 5 mM CaCl2 but in absence of P. carinii. Specific binding was defined as calcium-dependent binding and calculated by subtracting calcium-independent binding in presence of 5 mM EGTA from total binding. Calcium-independent binding in presence of 5 mM EGTA plus sedimentation of SP-A due to its self-association in presence of 5 mM CaCl2 constituted <25% of total binding. B: Scatchard analysis of specific-binding data from A. SP-ABound/SP-AFree, ratio of bound SP-A to unbound (free) SP-A. Normal SP-A binds to P. carinii with a dissociation constant (Kd) of 7.8 × 109 M and 7.1 × 106 SP-A binding sites/P. carinii organisms. TNM- and ONOO-treated SP-A binds to P. carinii with an increased Kd of 2.4 × 108 and 1.6 × 108 M, respectively, without affecting its no. of binding sites. Results are means ± SE of 3-6 experiments performed in duplicate. Some error bars are smaller than symbols.

Because P. carinii express a great abundance of surface mannose-type oligosaccharides (42, 43) and the specific affinity of SP-A for mannose has been well documented (15), -methyl-D-mannopyranoside was used to determine whether SP-A binds to P. carinii surface carbohydrate through its CRD. SP-A binding was inhibited by 72% in the presence of 750 mM -methyl-D-mannopyranoside (Fig. 4), which suggests that SP-A binds to P. carinii surface carbohydrate through its CRD.

View larger version (13K): [in this window] [in a new window]   Fig. 4.   Competitive inhibition of specific binding of SP-A to P. carinii by -methyl-D-mannopyranoside. Binding assays were performed with 1 × 106 P. carinii organisms and 100 ng of normal SP-A in presence of varying concentrations of -methyl-D-mannopyranoside in 100 µl of HBSS containing 1% BSA and 5 mM either CaCl2 or EGTA. Total binding was also corrected for nonspecific sedimentation of SP-A. Results are means ± SE of 4 experiments performed in duplicate. Significant difference compared with value in absence of competitor: * P < 0.05; ** P < 0.01.

Role of normal and ONOO-nitrated SP-A in P. carinii adherence and phagocytosis to alveolar macrophages.    Consistent with the findings of Williams et al. (60), the SP-A used in these adherence and phagocytosis studies was shown to enhance P. carinii adherence to alveolar macrophages in a concentration-dependent manner (Fig. 5A). However, for the first time, the results in Fig. 5A indicate that SP-A nitrated by ONOO significantly loses its ability to promote adherence of P. carinii to alveolar macrophages at 4°C (P < 0.01). Similarly, this difference was also demonstrated at 37°C where normal SP-A (10 µg/ml) enhanced adherence and phagocytosis of P. carinii by alveolar macrophages from 21.5 ± 2.0 to 36.7 ± 2.4% (P < 0.05). Adherence and phagocytosis were not significantly altered (18.3 ± 2.0%) by the presence of the same concentration of ONOO-nitrated SP-A (Fig. 5B).

View larger version (15K): [in this window] [in a new window]   View larger version (13K): [in this window] [in a new window]   Fig. 5.   Role of normal and nitrated SP-A in P. carinii adherence (A) or adherence and phagocytosis (B) to alveolar macrophages. A: 51Cr-labeled P. carinii were incubated with rat alveolar macrophages cultured in tissue culture plates in DMEM containing 0.1% BSA at 4°C for 4 h in absence and presence of either normal or ONOO-treated SP-A (0-20 µg/ml). P. carinii adherence to alveolar macrophages was enhanced in a concentration-dependent manner by normal SP-A but not by ONOO-treated SP-A. P. carinii adherence to alveolar macrophages was significantly lower in presence of ONOO-treated SP-A in comparison to normal SP-A at all concentrations tested. Results are expressed as means ± SE of 2 experiments performed in duplicate. Significant difference compared with values in presence of the same concentrations of normal SP-A, ** P < 0.01. B: 51Cr-labeled P. carinii were incubated with rat alveolar macrophages cultured in tissue culture plates in DMEM containing 0.1% BSA at 37°C for 2 h in absence (control) and presence of either normal or ONOO-treated SP-A (10 µg/ml). SP-A significantly enhanced adherence and phagocytosis of P. carinii by alveolar macrophages, whereas ONOO-treated SP-A lost its ability to mediate an interaction between P. carinii and alveolar macrophages. Significant difference (P < 0.05) compared with: * control; # value in presence of 10 µg/ml of normal SP-A.

SP-A binding to alveolar macrophages. Incubation of SP-A with macrophages revealed a biphasic binding curve with an inflection in the total binding curve at ~2.5 µg/ml. Nitration of SP-A by two doses of 0.5 mM ONOO did not significantly affect the binding of SP-A to macrophages, which also suggests that the binding between SP-A and macrophages is through the collagen-like region rather than through the CRD (Fig. 6).

View larger version (13K): [in this window] [in a new window]   Fig. 6.   Concentration-dependent binding of normal and ONOO-treated SP-A to alveolar macrophages at 4°C. Normal and ONOO-treated SP-A (0.5-15 µg/ml) were incubated with alveolar macrophages for 1 h at 4°C. Bound SP-A and total cell DNA/well were determined as described in METHODS. Nonspecific binding of SP-A to wells in absence of cells was also determined and only contributed <10% of total binding (data not shown). Values are means ± SE of 3 experiments performed in duplicate.

	DISCUSSION

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The data reported herein indicate that nitration of human SP-A decreases its binding affinity (i.e., increases the K_d) for P. carinii in a concentration-dependent fashion. Human SP-A contains eight tyrosine residues per monomer, which are located in its CRD (11). Nitration of one or more of these tyrosines decreases the acidic dissociation constant of tyrosine from 10 to 7.5 (49), rendering nitrotyrosine more hydrophilic, thus potentially inducing conformational change in the tertiary structure of the globular CRD region of SP-A secondary to alterations in its ionic charge. Previous studies by our laboratory (13, 19, 67) have shown that nitration of SP-A decreases its ability to aggregate lipids in the presence of calcium and to bind to mannose. Amino acid analysis of ONOO- or TNM-treated SP-A failed to identify any oxidized amino acids to account for these changes (13, 19). These results, along with the finding that the adherence of SP-A to P. carinii was competitively inhibited by -methyl-D-mannopyranoside, suggest that an intact CRD is necessary for the adherence of SP-A to P. carinii, in agreement with previous findings (69) showing that rat SP-A binds to P. carinii MSG through its CRD.

Levels of nitrotyrosine in control SP-A samples (0.001 mol nitrotyrosine/mol monomeric SP-A, which corresponds to 30 pmol nitrotyrosine/mg SP-A, assuming that the molecular mass of monomeric SP-A is 30 kDa) are similar to what has been measured by ELISA in normal rat lung tissue [~30 pmol nitrotyrosine/mg protein (51)], normal human serum albumin [~30 pmol nitrotyrosine/mg human serum albumin (24)], and normal human plasma low-density lipoprotein [~85 pmol nitrotyrosine/mg protein (24)]. Significantly higher nitrotyrosine levels have been measured in the lungs of pediatric patients who died with acute respiratory distress syndrome (18) and in the lungs of rats exposed to endotoxin (62) or hyperoxia.

SP-A may bind to alveolar macrophages by a number of different mechanisms. Because SP-A is a lectin as well as a glycoprotein with N-glycosidic glycans, it can bind to surface -D-mannosyl residues, C1q receptors, or SP-A receptors (5, 28, 41, 63, 64). Pison et al. (41) reported that the binding of SP-A to macrophages is blocked by collagen-like protein C1q and type V collagen in a dose-dependent fashion, also suggesting a collagen-like domain-mediated mechanism. However, another study (61) suggested that SP-A binds to alveolar macrophages through a mannose-dependent process that may involve the CRD of SP-A. Our results indicate that nitration of SP-A decreases its affinity for P. carinii but does not alter its binding to alveolar macrophages. Because nitration of SP-A decreases its ability to bind to mannose (19, 67), our findings suggest that, under our experimental conditions, binding of SP-A to macrophages is primarily mediated by its collagen-like domain, although the involvement of its CRD is not excluded (61).

Previous reports (10, 27, 53, 55, 63) suggested that SP-A and SP-D enhance the phagocytosis of bacteria and viruses. Indeed, in a recent report, Hickman-Davis et al. (21) demonstrated that the killing of Mycoplasma pulmonis by alveolar macrophages required the presence of SP-A. Williams et al. (60) reported that human SP-A enhanced adherence of P. carinii to alveolar macrophages. Our results clearly demonstrated that nitration of SP-A results in decreased binding to P. carinii and an abrogation of its ability to mediate P. carinii adherence to macrophages, whereas its binding to alveolar macrophages was not significantly affected. Because our present data and those of others (69) indicate that the CRD of the SP-A molecule interacts with the MSG on the P. carinii surface, whereas the collagen-like domain of the molecule interacts with alveolar macrophages, it is likely that nitration of SP-A interferes with its ability to serve as a ligand for P. carinii adherence to alveolar macrophages at the site of the SP-A molecule-P. carinii interaction rather than at the interaction between SP-A and macrophages. This conclusion is consistent with the speculation by Koziel et al. (26). However, they also found that human SP-A reduced rat P. carinii adherence to human alveolar macrophages by ~20%. Possible explanations for the discrepancy between the results of Koziel et al. (26) and ours include the use of rat versus human macrophages and the extent of control SP-A nitration.

This is the first report to show that the modification of a single amino acid in SP-A under pathological conditions modulates an important in vivo function, and thus our results are of much important biological significance. During lung inflammation, there is a marked upregulation of both · NO and O₂· production by alveolar macrophages (1, 14, 45, 48, 57, 63) in close proximity to SP-A. The reaction product of · NO and O₂· is ONOO, a potent oxidation and nitrating agent. A recent report (9) indicated that myeloperoxidase from infiltrated neutrophils in the lung also can catalyze chlorination and nitration reactions with nitrite (NO₂), the end product of · NO, and hydrogen peroxide, the dismutated product of O₂·, as substrates, suggesting a novel mechanism for protein nitration. A study by Zhu et al. (68) also showed that horseradish peroxidase catalyzes SP-A nitration with hydrogen peroxide and NO₂ as substrates and inhibits both its lipid aggregation and mannose-binding functions. Furthermore, our most recent studies (unpublished observations) show that reactive oxygen-nitrogen intermediates generated by rat alveolar macrophages stimulated by LPS nitrated human SP-A. This also suggests that, under inflammatory conditions, SP-A nitration may have a pivotal role in host defense of the lungs against P. carinii infection.

	ACKNOWLEDGEMENTS

We acknowledge the valuable comments and suggestions of Drs. John P. Crow and Imad Y. Haddad and the excellent technical assistance of Dr. Rajamouli Pasula and Carpantanto Myles.

	FOOTNOTES

This work was supported by National Heart, Lung, and Blood Institute Grants HL-31197 and HL-51173 (to S. Matalon) and HL-43524 and HL-51962 (to W. J. Martin II) and Office of Naval Research Grant N00014-97-1-0309 (to S. Matalon).

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.

Address for reprint requests: S. Matalon, Dept. of Anesthesiology, Univ. of Alabama at Birmingham, 619 South 19th St., Birmingham, AL 35233-6810.

Received 12 May 1998; accepted in final form 28 August 1998.

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