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Published ahead of print on September 4, 2003, doi:10.1165/rcmb.2003-0246OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 30, pp. 319-325, 2004
© 2004 American Thoracic Society
DOI: 10.1165/rcmb.2003-0246OC

Role of Surfactant Protein-A in Nitric Oxide Production and Mycoplasma Killing in Congenic C57BL/6 Mice

Judy M. Hickman-Davis, Julie Gibbs-Erwin, J. Russell Lindsey and Sadis Matalon

Departments of Anesthesiology, Genomics and Pathobiology, and Physiology and Biophysics, University of Alabama at Birmingham, Birmingham, Alabama

Address correspondence to: Dr. Sadis Matalon, Department of Anesthesiology, University of Alabama at Birmingham, UAB Dept. of Anesthesiology, 1530 3rd Ave. South, Birmingham, AL 35294-2172. E-mail: Sadis.Matalon{at}ccc.uab.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We generated congenic surfactant protein A (SP-A)–deficient (SP-A[-/-]) mice on the mycoplasma resistant C57BL/6 background (B6.SP-A[-/-]) and characterized their response to mycoplasma infection in comparison to C57BL/6 (B6) mice. B6.SP-A(-/-) mice infected with 106 colony-forming units (cfu) of Mycoplasma pulmonis had significantly higher bacterial lung loads than B6 mice at 72 h postinfection (p.i.). At the higher infection dose of 107, B6.SP-A(-/-) mice had significantly higher lung cfu at 24 h; however, no difference in mycoplasma cfu was observed between B6 and B6.SP-A(-/-) mice at 48 and 72 h p.i. We found that uninfected B6 mice had lower bronchoalveolar lavage nitrite (NO2-) and nitrate (NO3-) levels as compared with B6.SP-A(-/-) mice. On the other hand, infection of B6 mice with mycoplasmas resulted in significantly higher bronchoalveolar lavage NO2- and NO3- as compared with B6.SP-A(-/-) mice. These data indicate that SP-A may help regulate NO production in response to a specific stimulus, i.e., suppression of NO in the absence of bacteria and increased NO in the presence of bacteria. These data indicate that the contribution of SP-A to mycoplasma killing may be limited to lower doses of pathogens.

Abbreviations: alveolar macrophage, AM • acidophilic macrophage pneumonia, AMP • bronchoalveolar lavage, BAL • colony-forming unit, cfu • interferon-{gamma}, IFN-{gamma} • interleukin-1ß, IL-1ß • monocyte chemotactic protein, MCP • nitric oxide, NO • polymerase chain reaction, PCR • postinfection, p.i. • polymorphonuclear cells, PMN • surfactant protein A, SP-A • tumor necrosis factor-{alpha}, TNF-{alpha}


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Mycoplasma pulmonis infection in mice provides an excellent animal model of human respiratory mycoplasmosis. M. pulmonis infection in mice causes pneumonia with infiltration of airways with polymorphonuclear cells (PMNs) and alveoli with a mixture of activated PMNs and alveolar macrophages (AMs). In the absence of specific antibody, PMNs are unable to clear mycoplasmas (1) and appear to contribute more to the dissemination of organisms to other organs (2) and the development of lung pathology (3) than to resolution of disease. Mouse strains differ dramatically in resistance to infection with M. pulmonis, with C57BL/6 (B6) mice being highly resistant and C3H/He (C3H) mice being highly susceptible to disease (4). Our studies of innate host defenses against respiratory mycoplasmas have identified the AM as the primary effector cell in early mycoplasma killing in vivo (4) and in vitro (5). In vitro studies with activated AMs from mycoplasma-resistant B6 mice indicated that the pulmonary collectin surfactant protein A (SP-A) as well as nitric oxide (NO) were necessary for mycoplasma killing (5).

Previously, SP-A knockout mice on a mixed genetic background (129 x BS SP-A[-/-]) were used to determine the importance of SP-A for mycoplasma killing in vivo (6). These studies showed that SP-A(-/-) mice were significantly more susceptible to mycoplasma infection as compared with 129/J, mycoplasma-susceptible C3H, and mycoplasma-resistant B6 mice. However, because of the enormous contribution of mouse strain to disease severity (7), direct strain-matched comparisons are an absolute necessity to definitively determine the importance of SP-A for mycoplasma killing in vivo.

Accordingly, we backcrossed the available 129 x BS SP-A(-/-) mice onto the mycoplasma resistant B6 background and evaluated mycoplasma severity in the resulting congenic B6.SP-A(-/-) mice. Our findings indicate a complex role for in vivo regulation of NO by SP-A and indicate that the contribution of SP-A to mycoplasma killing may be limited to lower doses of pathogens.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Endotoxin-free phosphate-buffered saline and Dulbecco's modified Eagle's medium were from Cellgro (Atlanta, GA). Saline was from Abbott Laboratories (Abbott Park, IL). BBL mycoplasma broth base was from Becton Dickinson (Cockeysville, MD). Diff-Quik stain kits were from Baxter Healthcare (McGraw Park, IL). Unless stated, all other chemicals were from Sigma (St. Louis, MO).

SP-A
SP-A was purified from the bronchoalveolar lavages (BALs) of patients with alveolar proteinosis by n-butanol extraction as previously described (8). SP-A was tested for endotoxin, and only batches with < 0.5 endotoxin units/ml were used in experiments.

Animals
C57BL/6NCr (B6) mice were obtained from the National Cancer Institute, Frederick, MD. Breeding pairs of 129/Ola x Black Swiss SP-A–deficient (129 x BS SP-A[-/-]) mice were provided by Drs. Whitsett and Korfhagen (University of Cincinnati) and were bred in Trexler isolators. C57BL/6 SP-A(-/-) (B6.SP-A[-/-]) N10 mice were generated from 129 x BS SP-A(-/-) mice by backcrossing for 10 generations onto the B6 background. Genotype was determined using mouse tail DNA by polymerase chain reaction (PCR) with primers that identified the neomycin insert and the normal sp-a gene and confirmed by Southern blot analysis using the DNA probe created by Korfhagen and coworkers (9). Mice were maintained and health status monitored as previously described (3). All mice used in studies were 8–12 wk of age.

Western Blots
Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of SP-A samples was performed on BAL samples in a Bio-Rad Mini-Protein D slab gel apparatus (Bio-Rad, Cambridge, MA), under reducing conditions (5% mercaptoethanol). SP-A gels were transferred to nitrocellulose membranes using a Bio-Rad Mini-Tranblot Cell. Nonspecific protein binding to sites was blocked by incubation with 1% bovine serum albumin in Tris(hydroxymethyl)aminomethane (Tris)-Tween for at least 2 h. The nitrocellulose-bound antigen was overlaid with purified rabbit anti-human SP-A (1:10,000) kindly provided by Dr. Phelps (Hershey, PA). In control measurements the antibodies were replaced with rabbit nonspecific IgG. Bound antibody is detected with alkaline phosphatase–conjugated goat anti-rabbit immunoglobulin antibodies using 5-bromo-4-chloro-3-indolyl-1-phosphate and nitroblue tetrazolium kit (Bio-Rad).

Mycoplasmas
The UAB CT strain of M. pulmonis was used in all experiments. For in vivo experiments, 3 x 107 colony-forming units (cfu)/ml stock was diluted in broth A and inoculations were given intranasally in 50-µl volumes. cfus in inoculates were confirmed after serial dilution and plating (5).

Quantitative Lung Cultures
Mice were killed, and lungs were removed aseptically, individually minced, and sonicated for 1 min in broth A. Ten-fold serial dilutions were plated onto mycoplasma agar and the total number of cfus in the lungs of each animal was determined after incubation for 7 d.

BAL
BALs were collected as described previously (5). Briefly, mice were anesthetized and the proximal trachea exposed surgically. A sterile 19-gauge intravenous catheter was inserted through the wall 5 mm into the lumen of the trachea. For AM isolation, lungs were lavaged in situ with 20 separate 1-ml washes of sterile saline and lavagates from animals within groups were pooled and centrifuged to pellet the cellular fraction. Cells were > 90% viable by trypan blue exclusion and > 95% macrophages as differentiated on cytospin preps using Diff-Quik stain. For determination of cytokine, nitrite and nitrate and protein levels, lungs were lavaged with two separate 1-ml washes of sterile saline on ice. Individual lavage from each mouse was labeled, centrifuged to pellet the cellular fraction and the supernatant was removed and frozen at –80°C in 200-µl aliquots. Frozen lavage aliquots were thawed and used within 24 h or discarded; samples were not refrozen.

Protein Assay
Frozen aliquots of BAL samples were analyzed simultaneously by the Micro BCA*Protein Assay Reagent Kit (Pierce, Rockford, IL) using the microtiter plate protocol and a standard curve prepared from assaying known amounts of bovine serum albumin as per the manufacturer's instructions.

Nitrate and Nitrite
NO3- and NO-2 were measured by fluorescence utilizing 2,3-diaminonaphthalene (DAN) as previously described (8, 10). NO-3 was first converted to NO-2 with Escherichia coli reductase. One hundred microliters of sample was incubated in duplicate with 25 µl of freshly prepared DAN (0.05 mg/ml in 0.62 M HCL) for 10 min. The reaction was stopped by the addition of 2.8 N NaOH and the signal was measured using a fluorescent plate reader with excitation at 360 nm, emission at 450 nm and a gain setting of 100%. NO-2 concentration was determined using a NaNO2 standard.

Cytokines
Interferon (IFN)-{gamma}, interleukin (IL)-1ß, tumor necrosis factor (TNF)-{alpha}, and macrophage chemoattractant protein (MCP)-1 in frozen aliquots of BAL fluid were measured by Quantikine ELISA kits (R&D Systems, Minneapolis, MN) according to the manufacturer's instructions.

Pathology
Tissues were stained with hematoxylin and eosin (H&E). Lung sections were scored for mycoplasma lesion severity: (i) PMN exudate in airway lumina, (ii) hyperplasia-dysplasia of the mucosal epithelium, (iii) peribronchial and perivascular lymphoid accumulation, and (iv) parenchymal lesions (3, 11).

Statistics
All experiments had a minimum n of four samples per group. Unless otherwise stated, experiments were repeated at least once. Parametric data were analyzed by ANOVA followed by Tukey's multigroup comparison of the means after log conversion, or by Kruskal Wallis ANOVA and Pearson's correlation of the means for nonparametric data (Analytical Software, St. Paul, MN). P values of 0.05 or less were considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Generation of Congenic B6.SP-A(-/-) Mice
129 x BS SP-A(-/-) mice were backcrossed for 10 generations onto the B6 background. Tail biopsies were obtained from progeny at each generation for the isolation of genomic DNA to determine if the SP-A null mutation had been transmitted. Genotype was determined by PCR based on primers that identified the neomycin insert and the normal sp-a gene (Figure 1A) and confirmed by Southern blot analysis using the DNA probe created by Korfhagen and coworkers (7) (Figure 1B). Western blot analysis of BAL fluid from B6 mice demonstrated characteristic bands for SP-A at ~ 35 kD; these bands were absent in the BAL from B6.SP-A(-/-) mice (Figure 1C).



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  		Figure 1. Determination of B6.SP-A(-/-) N10 genotype. (A) PCR was performed on mouse tail genomic DNA using primers chosen from the printed amino acid sequence for SP-A to select for the neomycin insert (280 bp) in the SP-A(-/-) mice and flanking either side of an analogous portion of the normal sp-a gene (403 bp). (B) Mouse tail genomic DNA analyzed by Southern blot technique. DNA was probed with the 1.2-kb Hind III genomic mouse sp-a gene fragment to detect a 4.1-kb band of the normal allele and a 5.6-kb band of the targeted allele. (C) Western blot analysis was performed on BAL samples from B6 and B6.SP-A(-/-) mice. Wild-type (+/+), heterozygous (+/-), and homozygous (-/-) for the knockout are depicted below the lanes.

 
Pathology Associated with SP-A Genotype
Histologic changes characteristic of acidophilic macrophage pneumonia (AMP) (12, 13) were present in lungs of some SP-A(-/-) mice, particularly within the parent mixed strain 129 x BS SP-A(-/-) mice. Mild cases had only occasional AMs with intensely acidophilic cytoplasm containing very fine crystals scattered through alveoli, whereas severe cases had pooling of large acidophilic mono- to multi-nucleate AMs containing crystals of varying size (Figures 2A–2B). Occasionally, very large crystals were seen partially or completely extracellular in the air spaces. Prevalence and severity of these lesions tended to be age dependent. In mice < 6 mo of age the lesion occurred in 2/8 129 x BS SP-A(-/-), 0/8 B6.SP-A(-/-), and 0/9 B6. At 6–12 mo of age the lesion was present in 12/12 129 x BS SP-A(-/-) and 5/13 B6.SP-A(-/-) mice.



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  		Figure 2. Histopathologic changes in 129 x BS SP-A(-/-) mice. Lung section from an untreated 129 x BS SP-A(-/-) mouse stained with H&E. (A) Alveolar spaces are focally infiltrated with AMs containing brightly eosinophilic crystalline material (x200). (B) Magnification (x400) of alveolus from A (inset black box).

 
Effects of SP-A On Mycoplasma Infection In Vivo
We infected mice with 5 x 104, 3 x 106, and 1 x 107 cfu M. pulmonis and quantified cfu/ml in whole lung homogenates at 72 h postinfection (p.i.) B6.SP-A(-/-) mice had a 90% increase in mycoplasma numbers with the low infection dose (5 x 104) and significantly higher mycoplasma numbers (P = 0.02) at the moderate infection dose (3 x 106) as compared with B6 mice. However, at the high infection dose (1 x 107) there was no difference between B6.SP-A(-/-) and B6 mice (Figure 3).



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  		Figure 3. Mycoplasma killing dose response. B6 (filled bars) and B6.SP-A(-/-) (striped bars) mice were infected with M. pulmonis with 5 x 104, 3 x 106, or 1 x 107 cfus of M. pulmonis and killed at 72 h p.i. for determination of mycoplasmas in whole lung homogenates. *Significance from B6 mouse strain, P = 0.02. Results are means ± SE, 5 x 104 B6 n = 19, B6.SP-A(-/-) n = 16; 3 x 106 B6 n = 10, B6.SP-A(-/-) n = 11; 1 x 107 B6 n = 11, B6.SP-A(-/-) n = 11 mice per group. Graphed on a log scale.

 
In subsequent studies, mice were infected with 5 x 104, 3 x 106, and 1 x 107 cfu M. pulmonis, and lungs were examined for pathology 72 h p.i. Lungs were scored on the basis of severity of characteristic histopathologic lesions as defined in MATERIALS AND METHODS. No differences were detected between B6 and B6.SP-A(-/-) mice for any of the measured variables (Table 1) despite the presence of higher numbers of mycoplasmas within the lungs of B6.SP-A(-/-) mice.


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  		TABLE 1 Lung lesion pathology

 
We determined the time course of infection by infecting mice with a high dose of M. pulmonis (1 x 107 cfu) and quantifying mycoplasmas in whole lung homogenates at 24, 48, and 72 h p.i. B6.SP-A(-/-) mice had significantly higher levels of mycoplasmas (P = 0.02) as compared with B6 mice at 24 h p.i. However, there was no difference between B6 and B6.SP-A(-/-) mice at 48 and 72 h p.i. (Figure 4).



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  		Figure 4. Mycoplasma killing time course. B6 (filled bars) and B6.SP-A(-/-) (striped bars) mice were infected with 1 x 107 cfus of M. pulmonis and killed at 24, 48, or 72 h p.i. Mycoplasma cfu in whole lung homogenates, 24 h B6 n = 16, B6.SP-A(-/-) n = 19; 48 h B6 n = 13, B6.SP-A(-/-) n = 15; 72 h B6 n = 16, B6.SP-A(-/-) n = 19 mice per group. *Significance from B6 mouse strain, P = 0.02. Results are means ± SE. Graphed on a log scale.

 
As a further indication of damage to the alveolar epithelium, we measured total protein levels in BAL fluid from infected (1 x 107 cfu) mice at 24, 48, and 72 h p.i. BAL fluid protein levels were similar in B6 and B6.SP-A(-/-) mice, and did not change significantly regardless of infection status or time: uninfected B6 = 0.13 ± 0.01 mg/ml (n = 10), B6.SP-A(-/-) = 0.16 ± 0.03 mg/ml (n = 10); 24 h B6 = 0.27 ± 0.04 mg/ml (n = 8), B6.SP-A(-/-) = 0.19 ± 0.04 mg/ml (n = 8); 48 h B6 = 0.28 ± 0.16 mg/ml (n = 8), B6.SP-A(-/-) = 0.21 ± 0.06 mg/ml (n = 10); 72 h B6 = 0.21 ± 0.07 mg/ml (n = 8), B6.SP-A(-/-) = 0.27 ± 0.09 mg/ml (n = 8).

Effect of SP-A on NO Production
BAL fluid was collected from uninfected and 24, 48, and 72 h M. pulmonis (1 x 107 cfu)–infected mice for measurement of NO-2 and NO-3 levels. Uninfected B6 mice had significantly lower levels of NO-2 (after conversion of NO-3 to NO-2 as described in MATERIALS AND METHODS) (P = 0.03) as compared with uninfected B6.SP-A(-/-) mice. B6 and B6.SP-A(-/-) mice both had significant increases in NO-2 after infection with mycoplasmas; however, infected B6 mice had significantly higher levels of NO-2 (P < 0.02) in BAL fluid as compared with infected B6.SP-A(-/-) mice (Figure 5A). There was no change in NO-2 levels over time following infection. These data indicate that although SP-A decreases NO production in uninfected mice, it stimulates NO production following infection with mycoplasmas.



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  		Figure 5. NO production. (A) B6 and B6.SP-A(-/-) mice were infected with 1 x 107 cfus of M. pulmonis and killed at 24, 48, or 72 h p.i. for determination of NO in BAL fluid. *Significance from B6 mice within the same treatment group; {ddagger}significant difference from uninfected of the same strain; {dagger}significance from all other uninfected strains, B6 uninfected n = 8, B6.SP-A(-/-) uninfected n = 10; B6 24 h p.i. n = 8, B6.SP-A(-/-) 24 h p.i. n = 8; B6 48 h p.i. n = 8, B6.SP-A(-/-) 48 h p.i. n = 8; B6 72 h p.i. n = 21, B6.SP-A(-/-) 72 h p.i. n = 22 mice per group. (B) AMs isolated from B6 and B6.SP-A(-/-) mice and treated with IFN-{gamma}, SP-A or a combination of IFN-{gamma} plus SP-A. *Significance from media control of the same strain; {dagger}significance from all other strains and treatments, media B6 n = 42, B6.SP-A(-/-) n = 21; IFN-{gamma} B6 n = 39, B6.SP-A(-/-) n = 25; SP-A B6 n = 25, B6.SP-A(-/-) n = 22; IFN-{gamma} + SP-A B6 n = 25, B6.SP-A(-/-) n = 25 samples per treatment group. Results are means ± SE, P < 0.05.

 
AMs isolated from the BAL fluid of mice were treated with IFN-{gamma}, SP-A, or mycoplasmas to stimulate NO production. The addition of IFN-{gamma} upregulated NO-2 generation by AMs from B6 and B6.SP-A(-/-) mice, as compared with media alone. Treatment of AMs from B6 and B6.SP-A(-/-) mice with SP-A and the combination of IFN-{gamma} plus SP-A significantly upregulated NO-2 levels; however, AMs from B6 mice produced significantly more NO-2 in response to IFN-{gamma} plus SP-A as compared with B6.SP-A(-/-) mice (Figure 5B). Infection of AMs with mycoplasmas significantly increased NO-2 and NO-3 levels for both B6 and B6.SP-A(-/-) mice: 0.506 ± 0.08 and 0.383 ± 0.07 nmol NO-2 and NO-3 · 3 x 105 AMs-1 · h-1, n = 8 for both groups, respectively.

Effect of SP-A and Mycoplasmas on Cytokine Production
BAL fluid from infected and uninfected mice was tested for the presence of IFN-{gamma}, TNF-{alpha}, IL-1ß, and MCP-1 at 24, 48, and 72 h p.i. No differences in IFN-{gamma}, IL-1ß, or MCP-1 were observed between B6 and B6.SP-A(-/-) mice. However, B6.SP-A(-/-) mice had higher levels of TNF-{alpha} production at 72 h p.i. as compared with B6 mice (P = 0.025). Levels of both IL-1ß and MCP-1 were undetectable in the BAL fluid from uninfected mice (Table 2).


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  		TABLE 2 Cytokine production

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
SP-A–deficient mice produced by gene-targeted inactivation have been used to demonstrate a role for SP-A in pulmonary host defense against a number of bacterial and viral pathogens in vivo. In the past, pulmonary infection data generated with SP-A knockout mice has reported the background of these mice to be either on the inbred 129/J background (14–17), the outbred strain Black Swiss (18, 19) or on a mixed strain background (20). Previously, we used SP-A knockout mice to determine the role of SP-A in mycoplasma killing in vivo. These studies demonstrated that 129 x BS SP-A(-/-) mice were significantly more susceptible to respiratory mycoplasma infection than 129/J mice or mycoplasma-resistant B6 and -susceptible C3H mice, which represent both genetic extremes in response to infection (6). These data indicated that SP-A was important for mycoplasma killing in vivo. However, because wild-type littermate controls were unavailable to test this hypothesis, we backcrossed the 129 x BS SP-A(-/-) mice onto the B6 background to produce congenic B6.SP-A(-/-) mice (N10) to definitively determining the contribution of SP-A to mycoplasma killing in vivo.

There were a number of incidental pathologic findings in uninfected SP-A knockout mice. Routine histopathologic examination of tissues performed in the health maintenance of the breeding colony identified the development of AMP in 129 x BS SP-A(-/-) mice and to a lesser extent in B6. SP-A(-/-) mice. AMP is characterized by focal to widespread intra-alveolar accumulation of large acidophilic macrophages, whose cytoplasms are laden with crystalloid inclusions. Originally described as early as 1942 (21), this lesion has been identified primarily within the lungs of experimentally manipulated mice, and in genetically altered mice, particularly those generated on a B6 or 129/Sv background (13). We did not identify this lesion in any of the B6 mice examined; however, these mice were used at 8–12 wk of age. Previous reports of AMP in B6 mice indicate that this is an extremely rare occurrence (2/12,000 necropsies) and only then in aged mice (1.5–2 yr of age) (12). In mice with the motheaten (mev/mev) gene mutation, AMP is associated with macrophage dysregulation and pneumonia resulting in death (22). Recently, crystals were isolated from the BAL fluid of motheaten mice with AMP and identified biochemically as eosinophil chemotactic factor (Ym1), an interesting finding considering the lack of eosinophils within any of the described lesions (13). We do not believe that the formation of these crystals impacted any of our data involving B6 or B6.SP-A(-/-) mice as none of the mice examined developed AMP until after 6 mo of age and all mice were used in experiments at 2–3 mo of age. These incidental pathologic findings emphasize the importance for rederivation of these mice onto a congenic B6 background.

Previously we reported that pulmonary clearance of M. pulmonis was significantly decreased in 129 x BS SP-A(-/-) mice as compared with B6 mice for all time points and dosages examined. However, although there was a decreased ability of B6.SP-A(-/-) to clear mycoplasmas as compared with B6 controls, these differences were dose- and time-dependent. Interestingly, despite significant differences in bacterial lung loads, there were no significant differences in lung histopathology between B6 and B6.SP-A(-/-) mice. M. pulmonis causes a respiratory infection characterized by infiltration of the airways with PMNs, hyperplasia of airway epithelium, peribronchial and vascular lymphoid hyperplasia and infiltration, and infiltration of alveoli with AMs and PMNs. Mycoplasma lung disease severity generally correlates with numbers of M. pulmonis found within the lungs (23), and previous studies have monitored lung pathology in relation to genotype to identify specific genes or sets of genes that affect mycoplasma lung disease (24). The fact that increased bacterial numbers do not correlate with increased lung pathology in B6.SP-A(-/-) mice indicate that multiple genetic factors may contribute more to the cellular inflammatory response to mycoplasmas than either bacterial numbers or the presence of SP-A.

Because B6 mice are highly resistant to respiratory mycoplasma infection, we used a dose of mycoplasmas close to the LD50 (50% lethal dose) for B6 mice (25) to characterize the infection course in our new congenic strain. We found that differences between B6 and B6.SP-A(-/-) mice were transient. These data generated with matched strain B6 mice are analogous to previous studies performed with both Black Swiss SP-A(-/-) mice infected with Haemophilus influenzae (18) and 129/J SP-A(-/-) mice infected with Pseudomonas aeruginosa (14), where significant differences in bacterial clearance were only detectable at specific time points.

Previously, we demonstrated the importance of NO produced by AMs for mycoplasma killing in vivo (6). SP-A has been demonstrated to enhance NO production by AMs in vitro (26), although surprisingly, SP-A knockout mice have been shown to generate increased NO after infection with P. aeruginosa (14), group B streptococcus, and H. influenzae (18). We found that uninfected B6 mice had lower NO-2 levels in BAL fluid as compared with B6.SP-A(-/-) mice. However, B6 mice produced more NO-2 in response to mycoplasma infection than B6.SP-A(-/-) mice. These data support the idea that SP-A may at least in part help regulate NO production in response to a specific stimulus, i.e., suppression of NO in the absence of bacteria and increased NO in the presence of bacteria. These data are consistent with our previous data in which SP-A stimulated increased NO production from AMs isolated from transplant patients and inhibited NO production from AMs isolated from normal volunteers (8).

To further characterize the ability of SP-A and mycoplasmas to modulate NO production, we isolated AMs from B6 and B6.SP-A(-/-) mice and attempted to stimulate NO production in vitro. We were able to stimulate increased NO-2 production by AMs from both strains with either SP-A, IFN-{gamma} plus SP-A, or mycoplasmas as compared with untreated strain-matched AMs. In keeping with the in vivo data, B6 AMs produced significantly more NO-2 in response to IFN-{gamma} plus SP-A than AMs from B6.SP-A(-/-) mice. We found that B6 mice produced significantly greater amounts of NO as compared with B6.SP-A(-/-) mice, consistent with a role for NO in mycoplasma killing. In contrast, studies performed with Black Swiss SP-A(-/-) mice infected with group B streptococcus or H. influenzae and 129/J SP-A(-/-) mice infected with P. aeruginosa reported an increase in NO production by SP-A(-/-) mice as compared with controls (14, 18).

SP-A–deficient mice have been reported to have increased pulmonary inflammation (e.g., increased pathology as well as increased IFN-{gamma}, TNF-{alpha}, IL-1ß, IL-6) in response to infection with group B streptococcus, P. aeruginosa, H. influenzae, respiratory syncytial virus, adenovirus, and influenza A virus (6, 14–16, 20, 27). We found that by 72 h p.i. B6.SP-A(-/-) mice had increased levels of TNF-{alpha}, as compared with B6 mice; however, there were no differences in IL-1ß, IFN-{gamma}, or MCP-1. In vitro, SP-A has been reported to decrease TNF-{alpha} production in response to LPS (28) and peptidoglycan (29) by rat AMs and U937 cells. Likewise, SP-A was shown to downregulate not only TNF-{alpha} production but also TNF-{alpha} gene expression by Candida albicans–activated human AMs (30). Recently SP-A was demonstrated to inhibit TNF-{alpha} release by IFN-{gamma}– and IFN-{gamma} plus Mycobacterium avium–stimulated AMs from Swiss Webster mice (31). This latter study reported that SP-A inhibited NO production via a TNF-{alpha}– and NF-{kappa}B–dependent mechanism. These data do not fit with our findings in which B6 mice have higher NO production and yet lower TNF-{alpha} levels in response to mycoplasmas. However, differences in bacteria, experimental design (in vivo versus in vitro), and mouse strain must carefully be considered when comparing these data.

In summary, we have used SP-A knockout mice on the B6 background to definitively characterize the role of SP-A in mycoplasma infection in vivo. Although these mice demonstrated significant differences in reponse to respiratory mycoplasma infection, the dose- and time-dependent nature of these differences indicate that the importance of SP-A in mycoplasma killing may be limited to very early infection or to lower doses of pathogens. Interestingly, we found that SP-A was important for the regulation of NO production both in the basal state and in response to mycoplasma infection, and that this regulation did not appear to correlate with TNF-{alpha} activity. These data represent the first study to use congenic B6.SP-A knockout mice in demonstrating definitively a role for SP-A in mycoplasma clearance in vivo.


   Acknowledgments
 
The authors thank Dr. Ian Davis for expert advice, and Glenda Davis, Dr. Kedar Shrestha, Carpantato Myles, and Mark Phillips for technical support. This study was supported by grants from the NIH RR00149 (J.M.H.-D.), HL331197, and HL51173 (S.M.), and funds from the Veterans Affairs Research Service (to J.R.L.).

Received in original form June 26, 2003

Received in final form August 22, 2003


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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