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Published ahead of print on July 18, 2003, doi:10.1165/rcmb.2003-0091OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 30, pp. 271-279, 2004
© 2004 American Thoracic Society
DOI: 10.1165/rcmb.2003-0091OC

Surfactant Protein-D, a Mediator of Innate Lung Immunity, Alters the Products of Nitric Oxide Metabolism

Elena N. Atochina, Michael F. Beers, Samuel Hawgood, Francis Poulain, Christiana Davis, Trevor Fusaro and Andrew J. Gow

Pulmonary and Critical Care Division, University of Pennsylvania School of Medicine, Philadelphia; Children's Hospital of Philadelphia, Division of Neonatology, Philadelphia, Pennsylvania; and Cardiovascular Research Institute and Department of Pediatrics, University of California at San Francisco, San Francisco, California

Address correspondence to: Andrew J. Gow, Ph.D., Children's Hospital of Philadelphia, Abramson Research Center, Rm. 416A, 34th & Civic Center Blvd., Philadelphia, PA 19104. E-mail: Gow{at}email.chop.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 References
 
Surfactant protein (SP)-D, a 43-kD multifunctional collagen-like lectin, is synthesized and secreted by the airway epithelium. SP-D knockout (SP-D [-/-]) mice exhibit an increase in the number and size of airway macrophages, peribronchiolar inflammation, increases in metalloproteinase activity, and development of emphysema. Nitric oxide (NO) is involved in a variety of signaling processes, and because altered NO metabolism has been observed in inflammation, we hypothesized that alterations in its metabolism would underlie the proinflammatory state observed in SP-D deficiency. Examination of the bronchial alveolar lavage (BAL) from SP-D (-/-) mice reveals a significant increase in protein and phospholipid content and total cell count. NO production and inducible NO synthase expression were increased in the BAL; however, there was a decline in S-nitrosothiol (SNO) content in the BAL and a loss of SNO immunoreactivity within the tissue. This decline in SNO was accompanied by an increase in nitrotyrosine staining. We conclude that inflammation that occurs in SP-D deficiency results in an increase in NO production and a shift in the chemistry and targets of NO. We speculate that the proinflammatory response due to SP-D deficiency results, in part, from a disruption of NO-mediated signaling within the innate immune system.

Abbreviations: bronchiolar alveolar lavage, BAL • enzyme-linked immunosorbent assay, ELISA • inducible nitric oxide synthase, iNOS • large aggregate surfactant, LA • matrix metalloproteinase, MMP • nitric oxide, NO • small aggregate surfactant, SA • S-nitrosothiol, SNO • surfactant protein, SP


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 References
 
Surfactant protein (SP)-D is a member of a novel growing family of collagen-like lectins ("collectins") that are believed to play a role in non–antibody-mediated innate immune responses (1). SP-D shares considerable structural homology with other proteins of this type, including SP-A, conglutinin, bovine collectin-43, and mannose-binding protein. SP-A and SP-D are both multimeric Ca2+-binding lectins produced primarily by alveolar type II cells and nonciliated bronchiolar cells in the lung (2). In contrast to SP-A, SP-D does not interact with major surfactant phospholipids and is not associated with lamellar bodies or tubular myelin.

The primary function of these proteins appears to be in the modulation of host defense and inflammation, although the mechanism of their action has not been fully defined. In vitro, SP-A binds to specific cell surface receptors on alveolar macrophages (3) and type II epithelial cells (4), stimulates macrophage chemotaxis (5), and enhances the binding and killing of bacteria and viruses by alveolar macrophages (6). In vivo, SP-A deficiency leads to impairment in the clearance of selected classes of microbes, including Group B streptococci (7), Haemophilus influenzae (8), and Pneumocystis carinii (9). Recently it has been shown that isolated alveolar macrophages from SP-A (-/-) mice generated significantly less superoxide and hydrogen peroxide compared with wild-type alveolar macrophages (8). The ability of SP-A to modulate NO production by alveolar macrophages has been demonstrated with transformed cell lines (10) and primary rat alveolar macrophages (11). The importance of SP-A and NO for pulmonary clearance was shown in a mouse model of respiratory mycoplasmosis in which the production of both reactive nitrogen and oxygen species was required for myoplasmacidal activity (12).

Like SP-A, SP-D has been shown, in vitro, to stimulate neutrophil chemotaxis (13) and to bind to alveolar macrophages (14). In addition, it has been shown to bind and enhance the uptake of a variety of microbial species by macrophages, such as Escherichia coli, Mycobacterium tuberculosis, and P. carinii (15) and by neutrophils, including E. coli, Streptococcus pneumoniae, and Staphylococcus aureus (16). However, in contrast to SP-A, binding of SP-D does not correlate with phagocytosis of these organisms either in vitro or in vivo.

Whereas in vivo evidence appears to support a role for SP-A in the activation of cellular proinflammatory processes during clearance of organisms, targeted ablation of the SP-D gene results in an increase in the baseline level of inflammation. SP-D knockout (SP-D [-/-]) mice exhibit an increase in the number and size of macrophages within the airways, alterations in the surfactant phospholipid profile, increases in metalloproteinase activity, and early development of emphysema (17, 18). In addition, macrophages from SP-D (-/-) mice generate significantly greater levels of superoxide and hydrogen peroxide than those from wild type (8). To date, the relationship between SP-D, NO production and metabolism remains undefined.

NO possesses a well-recognized role in a number of physiologic processes; within the lung it is a regulator of both airway and vessel tone. It has been suggested that the mechanism of action of NO in the control pulmonary function is through S-nitrosylation, i.e., the formation of S-nitrosothiols (SNO) (19, 20). Furthermore, the balance of NO function between its physiologic and pathologic roles appears to be related to its interaction with other oxidants such that the predominant chemistry switches from nitrosylation to predominantly nitration, as evidenced by the formation of nitrotyrosine (21).

Given that NO and its metabolites have well-recognized roles during a variety of signaling processes, we hypothesized that the proinflammatory state of the lung in SP-D deficiency is associated with alterations in NO production and metabolism. In the present study we used the SP-D (-/-) mouse to demonstrate that SP-D deficiency results in alteration in the production and distribution of NO and its intermediates. The results presented here support a novel link between the lung collectins of the innate immune system and reactive nitrogen species.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 References
 
SP Antisera
The production and characterization of monospecific, polyclonal surfactant protein antisera against SP-A and SP-B have been previously described (22, 23). Anti–SP-A was produced in rabbits using purified rat SP-A and recognizes murine, bovine, and human SP-A. The polyclonal SP-B antibody was produced in rabbits using an organic extract of bovine surfactant (TA Surfactant; Ross Laboratories, Columbus, OH) as the immunizing antigen. This antisera crossreacts against rat, bovine, murine, and human forms of SP-B.

SP-D–Deficient Mice
SP-D (-/-) mice were generated by targeted gene inactivation as previously described (18). This was followed by backcrossing ten generations into C57BL/6 background. The lungs of SP-D (-/-) mice do not contain detectable SP-D mRNA or protein (data not shown). SP-D (-/-) mice survive and breed normally in the barrier facilities at the University of Pennsylvania. This colony, through extensive serologic and bacteriologic screenings, has been shown to be free of detectable viral or bacterial infection. Wild-type C57BL/6 littermates as well as mice purchased from Jackson Laboratories (Bar Harbor, ME) were used as controls. All mice were maintained under specific pathogen–free conditions. Experiments were performed between 8 and 12 wk of age on male and female mice. All experimental animals used in this study were under a protocol approved by the Institutional Animal Care and Use Committee of the University of Pennsylvania. Two groups of mice were compared: SP-D (+/+) mice (C57BL/6) and age-matched SP-D (-/-) mice with C57BL/6 genetic background.

Differential Cell Count and Surfactant Subfraction
Lungs of SP-D (+/+) and (-/-) mice were lavaged with 5 ml of sterile saline and total and differential cell counts were performed. Bronchoalveolar lavage (BAL) samples were centrifuged (400 x g for 10 min.) and the cell pellet was gently resuspended in 1 ml of phosphate-buffered saline for total cell count (Z1 Coulter counter; Beckman Coulter Inc., Fullerton, CA). The cells were spun on a Shandon Cytospin-3 preparation system at 750 rpm for 3 min and stained with a standard Kwik Diff (ThermoShandon, Pittsburgh, PA) for cell differentials or used for immunocytochemistry. Cells were identified as macrophages, eosinophils, neutrophils, and lymphocytes by standard morphology, and at least 200 cells were counted under x400 magnification. The percentage and absolute numbers of each cell type were then calculated. Immunocytochemical analysis was performed as previously described (24) using rabbit polyclonal anti-SNO sera (Calbiochem, San Diego, CA), a monoclonal antibody generated to nitrotyrosine (a gift from H. Ischiropoulos), or with a rabbit polyclonal antisera generated to mouse inducible NO synthase (iNOS; Calbiochem). The cell-free supernatant was separated into two fractions following a second centrifugation (20,000 x g for 60 min at 4°C). The resulting pellet contained the large-aggregate (LA) surfactant fraction. Although the supernatant contains soluble proteins as well as the small-aggregate (SA) fraction, for sake of simplicity and consistency with previous works (25) we termed the supernatant the SA fraction. The resulting LA pellets were resuspended in saline for biochemical characterization. The total protein content of the samples from LA and SA fractions was determined with the method of Bradford, with bovine IgG as a standard (26).

Phospholipid Assay
LA and SA surfactant fractions were analyzed for total phospholipid content by extraction of total phospholipids and determination of inorganic phosphorous content by the method of Bartlett (27).

Polyacrylamide Gel Electrophoresis and Immunoblotting
SDS-polyacrylamide gel electrophoresis of LA aggregate surfactant samples for SP-B was performed in 16.5% polyacrylamide gels using a Tris-Tricine buffer system as previously described (28) or for SP-A using NuPAGE 10% Bis-Tris gels (Novex, San Diego, CA) according to instructions of the manufacturer. Electrophoresed proteins (5 µg of total protein per each line) separated under reducing conditions were transferred to 0.2 µm nitrocellulose at 60 mA/cm2 for 12–18 h for subsequent immunoblotting or autoradiography. Western blots for SP-A and SP-B were performed as previously described (29). Immunoblotting was performed by incubation at room temperature in primary antibody for 1 h followed by incubation with goat anti-rabbit antisera conjugated to horseradish peroxidase (BioRad, Inc., Melville, NY). Specific proteins were visualized by enhanced chemiluminescence using the ECL Kit (Amersham, Inc., Arlington Heights, IL) and intensity of staining was analyzed by densitometric scanning of exposed film and quantitated using Quantity 1 (pdi Inc., Huntingdon Station, NY).

SP-A Enzyme-Linked Immunosorbent Assay
The SP-A content in the SA fractions was determined by using an enzyme-linked immunosorbent assay (ELISA) protocol with a commercially available kit (Vectastain 6100 kit; Vector Laboratories, Burlingame, CA). Aliquots of SA samples neat or diluted 1:2 with blocking buffer (1% fetal bovine serum in Dulbecco's phosphate-buffered saline) were applied to 96-well Nunc-Immuno MaxiSorp plates (Nalge Nunc International, Roskilde, Denmark). Each assay plate included a standard curve of human SP-A purified from patients with alveolar proteinosis (1–2,700 ng/well). Polyclonal anti–SP-A antiserum (PA3) was applied as a primary antibody (1:50,000) with goat anti-rabbit IgG (1:1,000) as the secondary antibody (Vectastain kit 6101; Vector Laboratories). Colorimetric detection of antibody binding was performed according to manufacturer's instructions and using TMB (TMB Substrate Reagent Set; BD PharMingen, San Diego, CA) as substrate. Color intensity was measured at 405 nm using a Bio-Rad Model 550 automated microplate reader (Bio-Rad, Hercules, CA) and analyzed with Bio-Rad Microplate Manager software, PC version 5.0.1. Values for unknown samples falling within the linear range of the standard curve were used to obtain the total SP-A content of each sample.

Lung Tissue Histology
After lavage, the right lung was inflated and fixed in paraformaldehyde (4% w/ Sodium Cacodylate 0.1M pH 7.3) for histologic analysis. Paraffin sections prepared from the lungs of SP-D (-/-) and (+/+) mice were stained with H&E for evaluation of airway inflammation. Sections were also used for immunohistochemistry for the detection of SNO and nitrotyrosine as previously described (24, 30). For the purposes of inflammation scoring, blinded slides were assessed as follows; 0, no lesions; 1, minimal lymphocytic restricted to perivascular and peribronchiolar regions; 2, moderate perivascular and peribronchiolar inflammation with mildly increased numbers of alveolar macrophages, lymphocytes, and eosinophils; 3, marked perivascular and peribronchiolar inflammation with moderately increased numbers of alveolar macrophages, lymphocytes, eosinophils, and multinucleated giant cells; 4, severe perivascular and peribronchiolar inflammation with markedly increased numbers of alveolar macrophages, eosinophils, lymphocytes, and multinucleated giant cells; 5, severe, perivascular, and peribronchiolar inflammation with effacement of alveolar parenchyma and small airways by sheets of inflammatory cells. For SNO and nitrotyrosine immunostaining scoring was performed on a scale of 0–4; 0 and 4 were representative of the negative and positive controls, respectively. Negative controls were treatment with organic mercury for SNO and reduction with sodium hydrosulfite for nitrotyrosine. Positive controls were treatment with acidified nitrite for SNO and with authentic peroxynitrite for nitrotyrosine. Details of these control treatments were as described previously (24, 30).

NO Measurements
The analysis of NO metabolites was performed by chemical reduction and chemiluminescence using the Ionics/Sievers Nitric Oxide Analyzer 280 (NOA 280; Ionics Instruments, Boulder, CO). Total nitrogen oxides were reduced by use of an excess of vanadium chloride in hydrochloric acid at 95°C. Due to the potency of this reduction system, it reduces all oxides of nitrogen in which the oxidation state of the nitrogen atom is higher than +2 including nitrate, nitrite, and SNO to release NO. Therefore, measurements produced using these conditions were considered as a total nitrogen oxide measurement. Both nitrite and SNO were measured independently. Nitrite analysis was performed using a potassium iodide and acetic acid mixture at room temperature, whereas SNO concentrations were measured using a copper cysteine method (31). Resultant signal areas from each assay were compared with standards to calculate the concentration of each nitrogen oxide. Sodium nitrate and nitrite (Sigma Chemical Co., St. Louis, MO) were used as the standards for the vanadium and iodide assays, respectively. SNO standards were generated by mixing equimolar amounts of acidified glutathione and nitrite in the presence of the chelator diethylenetriamine pentaacetic acid.

Data Analysis
Data were analyzed with the Sigma Stat standard statistical package (Jandel Scientific, Inc.. San Rafael, CA). Parametric data were analyzed with ANOVA or Student's t test assuming equal variances were performed to test differences between groups. Nonparametric data were analyzed by means of a Kruskal-Wallis test to test differences between groups. Correlations between data sets were analyzed by means of standard linear regression. Data are expressed as mean ± SEM. In all cases a P value of < 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
References
 
Ablation of the SP-D gene in mice results in persistent inflammation within the lung characterized by peribronchiolar mononuclear infiltrates, increased cell numbers in bronchoalveolar lavage, and early emphysematous changes in the parenchyma in association with an enhanced production of several proinflammatory mediators (17, 18). The results presented within this paper extend these observations with new studies demonstrating that an absence of SP-D protein in the airspace results in marked changes in NO production and metabolism. Within SP-D (-/-) mice, increases in iNOS expression and total NO production are accompanied by a significant alteration in NO metabolism characterized by enhanced production of higher oxides of nitrogen and accompanying decreases in generation of SNO. Our results represent the first report linking defects in the innate immune system in the lung with alterations in NO metabolism.

SP-D (-/-) mice have previously been generated by two separate groups using comparable transgenic technologies within NIH Swiss black (17) and CD-1 (18) genetic backgrounds. Within these studies we have used mice derived from one of these lines (18) backcrossed to the C57BL/6 strain. In agreement with previous studies there were no differences in either birth weight or mortality in SP-D (-/-) mice when compared with age-matched SP-D (+/+) mice on the C57BL/6 background (data not shown). Furthermore, SP-D (-/-) mice, within this background, possess perivascular and peribronchial infiltrates of mainly mononuclear cells within the lung parenchyma (Figure 1). The morphology of alveolar spaces was relatively preserved but contained foamy macrophages (Figure 1B). Histologic sections of SP-D (+/+) mice (Figure 1A) were uniformly normal and showed no evidence of lipoproteinosis or other abnormalities in lung morphology.



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  		Figure 1. SP-D deficiency resulted in parenchymal inflammation and significant airway neutrophilia. Representative paraffin-embedded, H&E-stained lung sections prepared from the left lung of SP-D (+/+) (A) and SP-D (-/-) mice (B). Sections were evaluated under light microscopy for inflammatory changes. Original magnification: x200. Inserts show BAL cell pellet under x600 magnification.

 
The BAL fluid of SP-D (-/-) mice showed a 2-fold increase in the number of macrophages compared with SP-D (+/+) mice (Figure 2). Macrophages accumulating in the lungs of SP-D (-/-) mice are grossly enlarged and often multinucleated (Figure 1B, inset). However, there also was an increase in the number of neutrophils and of lymphocytes in SP-D (-/-) mice (Figure 2). These observations lead one to conclude that SP-D ablation results in an activation of the pulmonary immune system. SP-D (-/-) mice were extensively evaluated for infection by culture and serology and did not show evidence of viral, bacterial, or fungal pathogens. This activation occurs in the absence of any pathogen, and thus presents a model system for the study of the effects of inflammation on the biochemistry of NO in the lung.



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  		Figure 2. SP-D deficiency alters the immune cell content of BAL. Absolute numbers of BAL cells were derived from counts in Giemsa-stained cytospin preparations and the total cell number in each BAL sample as described. Four cell types were counted: macrophages (MP), eosinophils (EP), neutrophils (NP), and leukocytes (LC). Data are expressed as mean ± SEM of n = 10 in the SP-D (+/+) (open bar) and n = 16 in the SP-D (-/-) mice (closed bar). *Significantly different from SP-D (+/+) value (P < 0.05).

 
Initially, we chose to characterize the effects of SP-D ablation in the C57BL/6 background on surfactant homoeostasis. SP-D (-/-) mice on both the NIH Swiss black (32) and CD-1 (18) backgrounds show marked increases in surfactant phospholipid content and SP-B protein content. To characterize the effects of SP-D ablation on surfactant homoeostasis within this strain, we examined the phospholipid and protein profiles of both LA and SA surfactant fractions (Table 1). In SP-D (+/+) mice the total phospholipid is apportioned equally between the LA and SA fractions. SP-D deficiency alters both the total amount and distribution of BAL phospholipid in LA and SA aggregate surfactant forms. Phospholipid content is increased within both the LA and SA fractions from SP-D (-/-) mice, reaching 188 ± 12% and 346 ± 25% of wild-type baseline levels, respectively. In SP-D (-/-) mice only 36 ± 1% of the total BAL phospholipid partitions with the LA fraction while 63 ± 4% is associated with SA surfactant, which is a significant redistribution from that observed in SP-D (+/+) mice. In contrast to phospholipid, analysis of total protein in BAL fractions of mice demonstrated that the majority of protein was found in SA fractions and was 2-fold increased by SP-D deficiency (Table 1). In accordance with the changes in phospholipid content, SP-B protein was specifically increased within the LA fraction by 180 ± 19%. Importantly, although LA fractions of SP-D (+/+) mice contain only 5–10% of total protein, SP-D deficiency did not alter these levels. Therefore, there are significant increases in both the level of SP-B and phospholipid content with SP-D ablation. These changes are similar to, but of lesser magnitude than, those seen in other strains.


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  		TABLE 1 Effect of SP-D deficiency on the phospholipid and protein content of BAL

 
To characterize expression of SP-A protein in this model the LA and SA aggregate fractions of SP-D (-/-) and (+/+) mice were analyzed by ELISA and Western blot. Using a polyclonal anti–SP-A antibody, the 35-kD monomeric SP-A protein was detected in LA surfactant fraction of SP-D (+/+) and SP-D (-/-) mice (Figure 3A). Group mean data obtained from densitometric scans of multiple samples of SP fractions indicated that SP-D deficiency significantly reduced, by 45%, the level of SP-A in LA fractions. This result was confirmed by ELISA analysis, which shows significant reduction of SP-A in the SA of SP-D (-/-) mice (Figure 3B). Similar changes in SP-A content have been noted in SP-D (-/-) mice on the Swiss black (32) but not on the CD-1 (18) background. The mechanism and physiologic relevance of the altered SP-A levels in the various SP-D (-/-) mice is unclear, but most likely represents strain variability. Of note, the relative levels of SP-A observed in the C57BL/6 strain used in this study (54% versus SP-D [+/+]; Figure 3) is comparable to that in heterozygous SP-A knockout mice, which do not show an enhanced inflammatory response or alterations in surfactant homeostasis. Therefore, it is unlikely that the changes observed in SP-D (-/-) mice can be attributed to alterations in SP-A expression.



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  		Figure 3. Effect of SP-D deficiency on SP-A protein expression. (A) Representative western blots performed sequentially with polyclonal antisera against SP-A visualized using enhanced chemiluminescence. Samples of LA surfactants prepared from BAL of SP-D (+/+) and SP-D (-/-) mice as described. 29–35 kD SP-A doublet bands in LA. Each lane contains 5 µg total protein. (B and C) Quantification of SP-A content within large (B) and small (C) aggregate fractions of the BAL. Densitometric scanning was performed from multiple blots and quantified as described in MATERIALS AND METHODS. Open bars: SP-D (+/+) mice; closed bars: SP-D (-/-) mice. Data are expressed as percent of SP-D (+/+) levels (B). SP-A content of the small aggregate fraction was assayed by ELISA as described in MATERIALS AND METHODS and is expressed as total content in ng (C). n = 13–15 samples were used in each group. Mean ± SEM was calculated after deriving the average of the results from three independent experiments. *Significantly different from SP-D (+/+) value (P < 0.05).

 
SP-D (-/-) mice show a temporal progression of postnatal airspace enlargement and spontaneous inflammatory changes. In other strains this inflammatory development has been reported to be associated with a progressive pulmonary emphysema, which is accompanied by increases in matrix metalloproteinase (MMP) activity and enhanced oxidant production by alveolar macrophages (17). Hydrogen peroxide formation by alveolar macrophages from SP-D (-/-) mice was increased 10-fold and was associated with enhanced expression of the metalloproteinases MMP-2, MMP-9, and MMP-12. The mechanism for these changes has not been defined. Within C57BL/6 mice we have documented a progressive increase in inflammatory markers although pulmonary emphysema has not been diagnosed.

Although SP-D (-/-) mice demonstrate increased oxygen radical production, the effects of these alterations on NO production and metabolism have not been considered in depth. Increased total nitrogen oxide production has been measured (a direct result of macrophage activation) (7); however, NO chemistry has not been characterized. Given the known effects of NO metabolites in inflammation, it is important to consider how SP-D deficiency affects NO biochemistry. Increased oxygen radical production should produce an alteration in NO chemistry, resulting in the increased formation of higher oxides of nitrogen, compounds in which the oxidation state of nitrogen is higher than +3, as evidenced by nitration or the formation of nitrate. Consequently, one would predict a decline in the production of compounds in which the oxidation state of the nitrogen is +2 or +3, the lower oxides of nitrogen, such as nitrite and SNO. Such a change in NO chemistry could result from either the direct interaction of NO with oxidants, such as superoxide, or from the oxidation of nitrite by oxidative enzymes such as eosinophil peroxidase and myloperoxidase. Therefore, the measure of total nitrogen oxides, both higher and lower oxides of nitrogen, relative to the lower oxidation state compounds, such as nitrite and SNO, may provide a useful measure for the involvement of oxidation in NO metabolism.

The production of NO metabolites was characterized in three major compartments of the lung: BAL, BAL cell pellet, and lung tissue. First, we examined the BAL for total nitrogen oxide, nitrite, and SNO content (Figure 4). Ablation of the SP-D gene results in a significant increase in total nitrogen oxide content without any significant change in nitrite, indicating a shift toward the formation of higher oxides of nitrogen. This shift is further demonstrated when one examines the yield of SNO within the BAL, which is significantly reduced in SP-D (-/-) mice. Autooxidation of NO produces equal quantities of nitrite and nitrate, and thus were this to be the predominant metabolic pathway for NO one would predict that nitrite would constitute 50% of the total nitrogen oxides, as is seen in SP-D (+/+) mice. Within SP-D (-/-) mice the balance of nitrogen oxide production is shifted away from lower oxide formation, as nitrite and SNO constitute only 22% of the total nitrogen oxide level. These observations indicate that the ablation of the SP-D gene results in a more oxidative environment. In agreement with this conclusion, lung tissue from SP-D (-/-) mice contains significantly higher levels of lipid peroxides and protein carbonyls (33).



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  		Figure 4. Yields of NO metabolites in BAL from SP-D (-/-) and (+/+) mice. BAL samples were analyzed by chemical reduction chemiluminesence for total NO, nitrite, and SNO as described in MATERIALS AND METHODS. Data are expressed as the total yield of NO within the lavage (A) or as a percentage of the total yield of nitrogen oxides from the lung lavage (B). Values are shown as mean ± SEM. Open bars represent SP-D (+/+) animals (n = 6), closed bars represent SP-D (-/-) animals (n = 5). *Significantly different from SP-D (+/+) value (P < 0.05).

 
Examination of BAL cell pellet showed that macrophages from the SP-D (-/-) mice appear to be in an activated state with a large and foamy cytoplasm (Figure 1B, insert). We, therefore, characterized NO metabolism in cell pellets from BAL of SP-D (+/+) and (-/-) mice by studying iNOS expression and SNO content (Figures 5 and 6). Examination of the BAL cell pellet for iNOS by western blot (Figure 5) shows that the iNOS content was significantly increased in SP-D (-/-) mice. This increase appears to result both from an increase in iNOS expression per cell (as represented by a 3-fold increase in the iNOS per µg protein) and from an increase in the total yield of material from the BAL (as shown by a 6-fold increase in the total protein content) (Figure 5B). In addition, immunocytochemical analysis reveals a shift in the intracellular localization of iNOS (Figures 6C and 6D). Within macrophages from SP-D (+/+) mice, iNOS is predominantly located in a tight perinuclear pattern, but within cells from SP-D (-/-) mice one sees that iNOS is distributed in a more diffuse punctuate pattern.



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  		Figure 5. Detection of iNOS within the BAL cell pellet samples of BAL cell lysate were analyzed by SDS-PAGE and Western blotting for the presence of iNOS. Staining was quantified as described and is expressed as percent of SP-D (+/+) level, either relative to protein content or as total yield. Values are expressed as mean ± SEM, n = 4, in each group. *Significant difference from SP-D (+/+), P < 0.05. Lung tissue samples were analyzed for iNOS content by immunohistochemistry; however, none was detected in either group.

 


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  		Figure 6. SP-D ablation results in cellular redistribution of iNOS and SNO immunocytochemistry of SNO (A and B) and iNOS (C and D) of BAL cell pellet of SP-D (+/+) (A and C) and SP-D (-/-) (B and D) mice. Cell pellets were prepared and stained for iNOS or SNO using polyclonal antisera and a FITC linked secondary as described. Representative images (original magnification: x200) are shown from five SP-D (+/+) and 6 SP-D (-/-) mice.

 
To examine whether the changes in iNOS distribution result in alterations in the production of SNO, we examined BAL cell pellet for SNO content by immunohistochemistry (Figure 6), using antibodies that have been extensively evaluated in a variety of systems (24). These data show a redistribution of SNO, which is similar to that observed for iNOS. Previously, it has been shown that RAW cell activation in culture is accompanied by an increase and a redistribution of SNO (24), and these data now provide an in vivo correlate of those findings. These alterations in SNO staining may represent either a consequence of increased and altered iNOS function or may reveal part of the NO dependent signaling pathway within macrophages. However, with the increased flux of NO generation accompanied by the increase in reactive oxygen species production that has been shown to occur in SP-D (-/-) macrophages, one would expect that higher oxide chemistry would prevail. This appears not to be the case as nitrotyrosine, a marker of higher oxide chemistry, is not significantly increased relative to SNO production in alveolar macrophages (data not shown). This implies that within the macrophage these reactive species are extremely tightly controlled, potentially as a mechanism to preserve the cell from damage.

In contrast to the changes seen within immune cells, immunohistochemical examination of the lung tissue from SP-D (+/+) and (-/-) mice revealed that there was a considerable shift toward higher oxide of NO metabolism (Figure 7). Within SP-D (+/+) mice SNO can be detected within both the small airways and blood vessels; however, nitrotyrosine appears to be below the sensitivity of detection. Ablation of the SP-D gene shifts this balance with a reduction in SNO staining and an increase in nitrotyrosine detection. These observations mirror those within the BAL, where SP-D ablation also resulted in a shift toward higher oxide chemistry. Both the degree of inflammation and the staining intensity of both SNO and nitrotyrosine were assessed by means of blinded nonparametric scoring (Table 2). As can be seen within the SP-D (-/-) mice, there is significant loss of SNO and gain of nitrotyrosine, whereas there is a significant rise in inflammation score.



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  		Figure 7. Immunohistochemistry of SNO and nitrotyrosine in lung tissues. Representative paraffin-embedded sections of lung tissue from SP-D (+/+) (A and B) and SP-D (-/-) (C and D) mice. Tissue sections were stained using either polyclonal antisera to SNO (A and C) or monoclonal antinitrotyrosine (B and D) and a horseradish peroxidase–linked secondary followed by staining with diaminobenzadine (as shown by brown stain). Sections were counterstained with H&E. Asterisks indicate areas positive for nitrotyrosine, whereas black arrows point to areas of SNO. The specificity of staining was confirmed using parallel sections which had been treated with p-chloromercuribenzoate (to remove SNO) or dithionite (to remove nitrotyrosine). Images are representative of eleven separate experiments (magnification: x200) in which nonparametric scoring indicated higher SNO staining in SP-D (+/+) and greater nitrotyrosine presence in SP-D (-/-). Median values for SP-D (+/+) are 3 for SNO and 0 for nitrotyrosine, whereas for SP-D (-/-) values are 1 for SNO and 1.5 for nitrotyrosine.

 

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  		TABLE 2 Effect of SP-D deficiency upon degree of inflammation and NO metabolite staining

 
The degree of inflammation observed within these mice has been shown to be variable. This variability was used to gauge how closely related the changes in NO chemistry were to the degree of inflammation (Table 3). Within the BAL, as one would predict, the macrophage content was closely correlated with iNOS expression. This observation indicates that not only are macrophages recruited to the BAL in SP-D ablation, but that these cells are activated. Despite an increase in total nitrogen oxide production, it was found that the yield of nitrite within the BAL was negatively correlated with macrophage content. Although this observation may be surprising, it is in agreement with other findings of this study that there is a shift in the products of NO metabolism toward higher oxide chemistry. Using the nonparametric data of the inflammation score and the ratings of staining intensity, the changes in NO-mediated protein modifications were assessed in relation to the degree of inflammation. In contrast to the increased production of NO metabolites within SP-D (-/-) mice, there was a significant negative correlation between inflammation and SNO staining. Nitrotyrosine, however, was positively correlated with inflammation. These findings further confirm that the inflammation, which occurs in response to SP-D ablation, is related with changes in NO metabolite chemistry.


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  		TABLE 3 Correlation between inflammation parameters and NO metabolite parameters

 
The appearance of nitrotyrosine and the loss of SNO within the lung seen in SP-D (-/-) mice may represent a "double hit" on lung function. The production of higher oxides of nitrogen has long been recognized to have potential toxic consequences, including DNA damage, lipid oxidation, protein oxidation, protein nitration, and cell death. However, NO, and in particular nitrosylation reactions such as the formation of SNO, has been shown to be critical in maintaining normal lung function such as the vasodilatation response (20). Therefore, the production of higher oxides of nitrogen represents not only the formation of potentially toxic compounds, but also the loss of NO that is required for normal pulmonary function. Furthermore, it is possible that the nonspecific production of such reactive intermediates adversely affects the capability of the pulmonary immune system. Indeed, preliminary studies have suggested that within SP-D (-/-) mice there is a reduced ability to clear Pneumocystis carinii following pulmonary infection (data not shown).

In summary, it would appear that ablation of the SP-D gene promotes an increase in baseline inflammation in the lung. Our studies are consistent with the concept that SP-D can operate as a potent regulator of the inflammatory system as evidenced by the marked changes in NO production, with a shift in the distribution and oxidative state of NO metabolites, observed in the lungs of SP-D (-/-) mice. In SP-D (-/-) mice there is a relative increase in the production of higher oxides of NO, and a concomitant reduction in nitrosylated products. A wide range of proteins have been identified to be nitrosylated both in vitro and in vivo (34). These proteins have been found to play a role in a variety of cellular processes; however, of particular interest has been the identification of nitrosylated proteins, which play a role in signal transduction pathways including: kinases, such as p21ras; channel proteins, such as the NMDA receptor; proteases, such as cathepsin B and the caspases; and transcription factors such as nuclear factor-{kappa}B (34). Therefore, it is conceivable that alterations in the degree of nitrosylation within the cellular milieu could represent a signaling mechanism by which SP-D achieves regulation of the inflammatory process. Particularly intriguing among these targets is nuclear factor-{kappa}B, which has been shown to be inhibited by S-nitrosylation of the p50 subunit within lung epithelial cell lines (35) and whose activity has been shown to be upregulated within alveolar macrophages from SP-D (-/-) mice (33). Furthermore, one can speculate that alterations in the nitrogen oxide chemistry of the lung underlie chronic pulmonary inflammation processes in general and as such may be critical in disease states such as asthma and Pneumocystis infection. Studies aimed at defining the pathways by which various nitrogen oxides may play a role in the regulation of pulmonary inflammatory processes are currently in progress.


   Acknowledgments
 
The authors thank Drs. A. Haczku and J. Casey for assistance with differential cell counts, Mr. Y. Tomer for expert technical assistance, Dr. H. Ischiropoulos for the generous gift of nitrotyrosine antibodies and, along with Dr. P. Ballard, assistance in the preparation of the manuscript. This research was supported by the NIH, HL-59867, and HL 64520 (M.F.B.); by the AHA (Scientist Development Grant to A.J.G.); and by the CROWN Foundation (A.J.G.).

Received in original form March 19, 2003

Received in final form June 27, 2003


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 References
 

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