Nitric Oxide Mediates Relative Airway ... - ATS Journals

Nitric Oxide Mediates Relative Airway Hyporesponsiveness to Lipopolysaccharide in Surfactant Protein A–Deficient Mice Amy M. Pastva1, Julia K. L. Walker1, Lee A. Maddox1, Sambuddho Mukherjee1, Charles Giamberardino1, Bethany Hsia1, Erin Potts1, Hongmei Zhu1, Simone Degan1, Mary E. Sunday1, Barbara L. Lawson1, Thomas R. Korfhagen2, David A. Schwartz3, Jerry P. Eu1, William M. Foster1, Timothy J. McMahon1, Loretta Que1, and Jo Rae Wright1 1 Duke University Medical Center, Durham, North Carolina; 2Children’s Hospital Medical Center, Cincinnati, Ohio; and 3National Jewish Medical and Research Center, Denver, Colorado

Surfactant protein A (SP-A) mediates innate immune cell responses to LPS, a cell wall component of gram-negative bacteria that is found ubiquitously in the environment and is associated with adverse health effects. Inhaled LPS induces lung inflammation and increases airway responsiveness (AR). However, the role of SP-A in mediating LPS-induced AR is not well-defined. Nitric oxide (NO) is described as a potent bronchodilator, and previous studies showed that SP-A modulates the LPS-induced production of NO. Hence, we tested the hypothesis that increased AR, observed in response to aerosolized LPS exposure, would be significantly reduced in an SP-A–deficient condition. Wild-type (WT ) and SP-A null (SP-A2/2) mice were challenged with aerosolized LPS. Results indicate that despite similar inflammatory indices, LPS-treated SP-A2/2 mice had attenuated AR after methacholine challenge, compared with WT mice. The attenuated AR could not be attributed to inherent differences in SP-D concentrations or airway smooth muscle contractile and relaxation properties, because these measures were similar between WT and SP-A2/2 mice. LPS-treated SP-A2/2 mice, however, had elevated nitrite concentrations, inducible nitric oxide synthase (iNOS) expression, and NOS activity in their lungs. Moreover, the administration of the iNOS-specific inhibitor 1400W completely abrogated the attenuated AR. Thus, when exposed to aerosolized LPS, SP-A2/2 mice demonstrate a relative airway hyporesponsiveness that appears to be mediated at least partly via an iNOS-dependent mechanism. These findings may have clinical significance, because recent studies reported associations between surfactant protein polymorphisms and a variety of lung diseases. Keywords: surfactant protein A; lipopolysaccharide; airway responsiveness

The epithelium of the lung is one of the largest interfaces between the body and the environment, and it is constantly exposed to air laden with a broad spectrum of bacteria, viruses, fungi, and allergens. The pulmonary host defense system must ignore innocuous antigens and, at the same time, effectively clear microbial pathogens, while minimizing the harmful consequences of the response that can result in excessive inflammation, tissue damage, and chronic lung disease. Surfactant(Received in original form August 6, 2009 and in final form March 5, 2010) This work was supported by National Institutes of Health grants 1K08-AI068822 (A.M.P.), 5P50HL084917 (J.R.W., W.M.F., A.M.P., E.P., B.H., S.M., C.G., S.D., and M.E.S.), 5R01HL068072 (J.R.W. and L.A.M.), 5R01HL058795 ( T.R.K.), 1P30ES011961 (D.A.S., J.R.W., and W.M.F.), 5R01HL084123 ( J.K.L.W.), VAMC ( J.K.L.W.), 5R01HL081285 ( J.P.E.), 5R01HL086887–03 (L.Q.), and HL-079915 ( T.J.M.). Correspondence and requests for reprints should be addressed to Amy M. Pastva, P.T., Ph.D., Duke University Medical Center, Box 3709, Durham, NC 27710. E-mail: [email protected] This article has an online supplement, which is accessible from this issue’s table of contents at www.atsjournals.org Am J Respir Cell Mol Biol Vol 44. pp 175–184, 2011 Originally Published in Press as DOI: 10.1165/rcmb.2009-0284OC on March 26, 2010 Internet address: www.atsjournals.org

CLINICAL RELEVANCE Our group and others indicated a role for surfactant protein A (SP-A) in regulating various inflammatory responses. Our new data further demonstrate a role for SP-A in altering a very significant physiologic component of many lung diseases, airway responsiveness (AR), and indicate a possible mechanism involving the regulation of inducible nitric oxide synthase by SP-A. Our findings show for the first time that a disconnect exists between inflammation and AR that is mediated by SP-A. These findings may have clinical significance, because recent studies reported associations between surfactant protein polymorphisms and a variety of lung diseases.

associated protein (SP)–A and SP-D are integral components of the pulmonary host defense system. They are members of a family of innate immune effector proteins known as collectins that are defined by their N-terminal collagen-like regions and globular C-terminal lectin domains. The lectin domains bind to a variety of carbohydrate moieties commonly found on microorganisms. Through this interaction, both SP-A and SP-D promote the clearance of invading pathogens by acting as opsonins to enhance microbe clearance, and by modulating the immune cell production of free radicals and free radical–mediated killing (1). A role for SP-A in mediating lung immune responses is supported by studies with SP-A–deficient (SP-A2/2) mice. Although these mice lack the delicate tubular myelin structures seen in wild-type (WT) mice, they exhibit no differences under normal physiologic conditions in lung morphology, alveolar phospholipid pool size and composition, or lung compliance and function (2). However, in response to challenges of intratracheally instilled Pseudomonas aeruginosa, Group B Streptococcus, or respiratory syncytial virus, SP-A2/2 mice demonstrate decreased microbe clearance from the alveolar space, increased inflammatory cell infiltrates, and increased tissue markers of inflammation (3–5). Thus, SP-A2/2 mice demonstrate substantial defects in host defense responses to both bacteria and viruses. In addition to modulating immune cell responses to pathogens, SP-A also mediates responses to endotoxin or LPS, which is the major cell-wall component of gram-negative bacteria. LPS, found ubiquitously in the environment, was shown to contribute substantially to the pathophysiology of lung diseases, including pneumonia, acute lung injury, and acute respiratory distress syndrome (6). Furthermore, exposure to air pollution and, more specifically, particulate matter (PM), of which LPS is a soluble component, is associated with adverse health effects and is linked with increased hospital admissions, use of medi-

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cations, acute respiratory symptoms, and transient decreases in pulmonary function, especially in those with known cardiovascular and pulmonary conditions (7–9). Epidemiologic data also indicate that LPS is important in the development and modulation of human asthma (10). Inhaled LPS activates the Toll-like receptor 4 (TLR4) signaling pathway, resulting in neutrophilic lung inflammation, increased airway responsiveness (AR) (11), and the production of inflammatory cytokines and reactive species such as nitric oxide (NO) (12, 13). SP-A, which is both aptly located and functionally adapted to interact with LPS inhaled from the environment, was shown to inhibit the LPS-mediated production of NO and Th1 cytokines by alveolar macrophages both in vitro and in vivo. SP-A2/2 mice challenged with intratracheal LPS in general have higher lung lavage fluid concentrations of NO and inflammatory cytokines than do WT mice, and when SP-A is restored by intratracheal administration, the LPSinduced production of NO in lung lavage fluid is reduced to concentrations comparable to those in WT mice (14). The inhibition of NO production by SP-A occurs via a variety of mechanisms, including the inhibition of LPS binding to the LPS binding protein (15), physical interactions of SP-A with the lipid A moiety on the rough LPS chemotype (16), and interactions of SP-A with CD14, a cell surface protein expressed on macrophages that acts as a coreceptor with TLR4 and myeloid differentiation protein-2 for the detection of LPS (17). A CD14-independent pathway of the SP-A–mediated inhibition of LPS-stimulated responses by alveolar macrophages was also identified (18). A perturbation in AR constitutes a significant physiologic component of many lung diseases. Despite intensive research, the mechanisms underlying both acute and chronic airway hyperresponsiveness are not well-defined. Numerous interactive processes appear to be involved, and an explicit examination and compilation of cellular and molecular mechanisms are imperative. A recent report by Garantziotis and colleagues, using a relevant inhalational challenge, linked innate immunity to the development of increased AR, and added further support to the hypothesis that endogenous ligands of TLR4 act as innate immune activators in environmental lung injury (19). In addition, a single direct delivery of aerosolized endotoxin to epithelial surfaces was shown to induce airflow obstruction that commences within minutes of challenge, can persist for up to 48 hours, and is highly variable in terms of absolute severity of the response (20). However, the mechanisms that regulate the development and severity of physiologic responses to LPS still remain unclear. Our current study is the first to investigate the role of SP-A in LPS-induced pulmonary inflammation and AR. Taken together, our results develop a novel paradigm to explain the manner in which prototypal pulmonary inflammation induced by the inhalation of LPS is dependent on SP-A. We observed that the robust airway inflammation from exposure to LPS coexisted simultaneously with airway patency and homeostasis of airway mechanical function. Although our conclusion remains speculative, this finding associates the lung collectins (specifically SP-A) with the reported variability in the physiologic response of airways to LPS.

MATERIALS AND METHODS Detailed methods are described in the online supplement.

Animal Exposures Ten-week-old to 12-week-old WT and SP-A2/2 mice (C57BL/6) were used in these studies. Aerosolized LPS (O111:B4 Escherichia coli LPS; Sigma, St. Louis, MO) or saline was administered for 2 hours. In some experiments, mice received 100 mg/kg intraperitoneally of the specific

inducible nitric oxide synthase (iNOS) inhibitor, 1400W (Caymen, Ann Arbor, MI), 20 minutes before exposure to LPS (21). Additional doses of 50 mg/kg were given 2 and 4 hours after the initial dose. Vehicle consisted of intraperitoneal saline.

Respiratory System Physiologic Measurements Measurements obtained from a flexiVent (Scireq, Montreal, QC) (22) were averaged after each methacholine (Mch) dose, and graphed as fold change from the average baseline measurement.

Lung Samples Bronchoalveolar lavage fluid (BALF) cell differentials were determined on at least 300 cells, using standard morphologic criteria. BALF total protein concentrations were determined using bicinchoninic acid (Pierce, Rockford, IL), and TNF-a and keratinocyte chemoattractant (KC) protein concentrations were analyzed via ELISA (R&D Systems, Minneapolis, MN). BALF SP-D concentrations were analyzed by Western blot analysis, using a GE Typhoon and ImageQuant software (GE Life Sciences, Piscataway, NJ). Perfused lung tissue was either flash-frozen for later protein analyses, or digested and prepped for RNA or flow cytometric analyses. Formalinfixed sections of lung tissue were stained with hematoxylin and eosin, and the extent of leukocyte inflammation was subjectively assessed after random coding.

Airway Smooth Muscle Measurements Main-stem bronchi were isolated, and ex vivo airway smooth muscle (ASM) responses were evaluated as described by Du and colleagues (23), with modifications. The tension after the addition of carbachol and isoproterenol is graphed as a percentage of the maximum tension induced by 3 mM carbachol.

NO Derivatives BALF nitrite was measured using the Griess assay according to Schmidt and colleagues (24), with modifications. RT-PCR was performed on RNA isolated from lung digest cells using primers for iNOS with cyclophilin as basal control. The iNOS/cyclophilin values from WT sham control samples were averaged, and each sample from the remaining groups was compared with this average value to obtain a relative fold change over WT sham control. Flow cytometric analysis of iNOS expression was performed using lung digest cells. Nitric oxide synthase activity was measured using the NOSdetect assay kit (Alexis KI01, Enzo Life Sciences International, Inc., Plymouth Meeting, PA). This assay measures the NOS-catalyzed conversion of radioactive arginine to citrulline. Data are expressed as fold difference compared with WT sham controls. S-nitrosothiol (SNO) was quantified in freshly homogenized perfused mouse lung tissue by mercury-coupled, photolysis–chemiluminescence detection, as previously described (25).

Statistical Analyses Statistical analyses were performed using Prism 4.0b (GraphPad Software, La Jolla, CA). Values are presented as means 6 SEM. A repeated-measures ANOVA with the Bonferroni post hoc test was used to determine differences in AR and ASM responses. Multiple group comparisons were made using ANOVA with the Tukey post hoc test to determine which group means differed. When two group comparisons were made, the Student t test was used. For all tests, P < 0.05 was considered statistically significant.

RESULTS SP-A–Deficient Mice Exposed to LPS Had Decreased Methacholine-Induced AR Compared with WT Counterparts

Multiple parameters, as measured by flexiVent technology, were used to assess alterations in respiratory system physiologic function and AR. The overall measure of total respiratory system resistance is designated RT (cm H2O/ml/second). Newtonian resistance (RN, cm H2O/second/ml) represents the resistance of the conducting or central pulmonary airways. Tissue damping (G, cm H2O/ml) reflects the viscous dissipation of energy in tissues,

Pastva, Walker, Maddox, et al.: SP-A–Deficient Mice Have Hyporesponsive Airways

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TABLE 1. RESPIRATORY SYSTEM PHYSIOLOGIC MEASUREMENTS DID NOT DIFFER SIGNIFICANTLY AT BASELINE (0 mg/mg METHACHOLINE) AMONG GROUPS OF MICE WT Sham RT RN G H C

0.51 0.24 4.31 25.69 0.044

6 6 6 6 6

0.06 0.02 0.53 1.54 0.003

SP-A2/2 Sham 0.57 0.26 4.23 23.24 0.048

6 6 6 6 6

0.02 0.03 0.27 2.78 0.003

WT LPS 0.60 0.22 4.38 24.85 0.037

6 6 6 6 6

0.05 0.04 0.32 0.37 0.001

SP-A2/2 LPS 0.53 0.30 4.37 24.85 0.037

6 6 6 6 6

0.04 0.05 0.23 2.52 0.003

WT Sham 1400W 0.56 0.27 5.29 25.07 0.039

6 6 6 6 6

0.02 0.01 0.28 0.72 0.001

SP-A2/2 Sham 1400W 0.57 0.31 4.55 23.59 0.039

6 6 6 6 6

0.03 0.03 0.33 1.60 0.002

WT LPS 1400W 0.59 0.26 5.03 25.84 0.040

6 6 6 6 6

0.03 0.02 0.38 2.41 0.003

SP-A2/2 LPS 1400W 0.55 0.36 4.65 22.05 0.040

6 6 6 6 6

0.02 0.01 0.26 0.88 0.003

Definitions of abbreviations: WT, wild-type; SP-A, surfactant protein–A; RT, total respiratory system resistance (cm H2O/ml/second); RN, central airway resistance (cm H2O/second/ml); G, tissue damping (cm H2O/ml); H, tissue elastance (cm H2O/ml); C, compliance (ml/cm H2O).

and represents parenchymal tissue resistance and airflow heterogeneity. Tissue elastance (H, cm H2O/ml) reflects the elastic energy storage in tissues, and was shown to represent airway closure (22). Respiratory system compliance (C, ml/ cm H2O) represents the ease with which the lungs can be inflated. Baseline measurements of RT, RN, G, H, and C (Table 1) and resting tracheal pressure were no different among the murine groups before any dose of MCh (data not shown). Sham-exposed WT and SP-A2/2 mice displayed similar MCh dose–dependent increases in AR (Figure 1), because RT, RN, G, and H increased minimally with increasing doses of Mch. As expected, exposure to aerosolized LPS caused a significant enhancement of AR in both WT and SP-A2/2 mice. However, the increased AR was significantly less pronounced in SP-A2/2 mice relative to WT mice. More specifically, RT, RN, G, and H increased with each increasing dose of Mch in WT mice. In SP-A2/2 mice, however, RN (i.e., resistance in the central airways) appeared to account primarily for the increased AR (RT), because levels of G and H were similar to those of sham mice (Figure 1). WT and SP-A–Deficient Mice Exhibited Classic LPS-Associated Inflammatory Responses

Inhaled LPS exposure is associated with lung neutrophilia, increased total protein concentrations, and increased concentrations of Th1-associated cytokines. As expected, the total cell and neutrophil counts, total protein concentrations, and tumor necrosis factor a (TNF-a) and KC protein concentrations rose substantially in the BALF of LPS-treated WT and SP-A2/2 mice relative to sham mice. Both the WT and SP-A2/2 LPStreated groups had similar responses (Figures 2A–2C). Normal lung histology was observed in the absence of LPS treatment (data not shown). LPS treatment resulted in robust neutrophilic infiltration, to a similar extent between the two LPS-treated groups (Figures 3A–D). These data suggest that the suppression of AR in LPS-treated SP-A2/2 mice is not a consequence of differences in classic LPS-associated inflammatory responses relative to WT counterparts. SP-D Concentrations Increased Similarly in LPS-Treated WT and SP-A–Deficient Mice

SP-D is another surfactant-associated protein that has potent immunomodulatory capabilities. However, unlike SP-A, the role of SP-D in AR is more clearly elucidated. Elevations in SP-D protein concentrations that occur with allergen challenge were shown to correlate with the extent and kinetics of inflammatory cell influx, inflammatory cytokine production, and AR (26). In our study, levels of SP-D expression, which did not differ in sham-treated mice, increased to similar degrees in LPS-treated WT and SP-A2/2 mice (Figure 4), suggesting that the extent of inflammatory response and suppression of AR in LPS-treated SP-A2/2 mice is not a consequence of differences in levels of SP-D expression.

ASM Contractile and Relaxation Responses Were Similar between WT and SP-A–Deficient Mice

Because impaired ASM relaxation, heightened ASM contractile responses, and alterations in ASM receptor signaling were all described as mechanisms in the development of increased AR (21, 27, 28), we isolated bronchial rings from WT and SP-A2/2 mice and compared their ASM properties (Figure 5). The rings were placed in a tissue bath and precontracted with KCl, which acts through voltage-gated L-type channels and therefore represents receptor-independent contraction. We found no difference between the maximum contraction recorded after KCl treatment in WT and SP-A2/2 mice, indicating no overall defect in voltage-gated smooth muscle contraction (data not shown). To rule out the possibility that a defect in muscarinic receptor– induced contraction is responsible for the attenuated AR observed in SP-A2/2 mice, increasing doses of carbachol were added to the bath to mimic the receptor-mediated contraction that would be expected during in vivo MCh challenge. We found no difference between groups in their contractile response to carbachol (Figure 5A) or in the maximum carbacholinduced tension (Figure 5, inset). Finally, to investigate the relaxation potential of the ASM from WT and SP-A2/2 mice, increasing doses of the b-adrenergic receptor agonist isoproterenol were added to induce relaxation. Again, we found no differences between groups (Figure 5B). Thus, the suppression of AR in SP-A2/2 mice is not a consequence of inherent differences in ASM contractile or relaxation responses. Concentrations of NO Derivatives Were Elevated in SP-A–Deficient Mice

NO is produced in a series of reactions that are catalyzed by one of three isoforms of the enzyme NOS. Both constitutive NOSs (neuronal/NOS1 and endothelial/NOS3) are calcium-dependent and generate NO within seconds. In contrast, inducible NOS (iNOS/NOS2) is calcium-independent and requires cytokines or LPS stimulation to be expressed, and maximal induction occurs a few hours after stimulation. Moreover, because previous studies showed that SP-A inhibits the LPS-induced production of NO both in vitro and in vivo (12–14), we quantified BALF nitrite concentrations and lung iNOS mRNA and protein expression, NOS enzymatic activity levels, and SNO concentrations. NO reacts with a variety of targets to mediate its effects, and the reactive pathway followed by NO is determined by the biochemical characteristics of the particular tissue environment. For example, NO reacts with oxygen in solution to form nitrite, a relatively stable and nonvolatile breakdown product. Thus, we quantified nitrite concentrations in the BALF. The amount of nitrite was greater in the BALF of SP-A2/2 mice compared with WT mice after either sham or LPS treatment (Figure 6A), in accordance with the results of previous studies using either intratracheal LPS or bacterial challenge (5, 14). Because stimulation with LPS induces the expression of iNOS, single-cell suspensions from whole-lung cell digests were sub-

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Figure 1. Surfactant protein A (SP-A)2/2 mice exposed to LPS had relatively hyporesponsive airways. Respiratory system physiologic measurements, including total lung resistance (RT), central airway resistance (RN), tissue damping (G), and tissue elastance (H ) and compliance (C ), were acquired after aerosolized methacholine (Mch) challenge (0 mg/ml, 10 mg/ml, 25 mg/ml, and 100 mg/ml). (A–D) No differences were evident between shamtreated wild-type (WT ) and SP-A2/2 mice at any dose of Mch. SP-A2/2 mice exposed to LPS (dotted line) were hyporesponsive compared with WT mice exposed to LPS (dashed line). The average of each physiologic measurement per Mch dose was graphed as fold change from baseline measurement. (n 5 6–12 mice/group; *P < 0.05, comparing LPS-treated mice with sham-treated mice; ^P < 0.05, comparing LPS-treated WT mice with LPS-treated SP-A2/2 mice).

jected to RT-PCR and flow cytometric analyses for iNOS message and protein concentrations, respectively. Cells from LPStreated SP-A2/2 mice exhibited the greatest concentrations of iNOS mRNA and protein expression (Figures 6B and 6C). Although a trend apparently exists for increased iNOS mRNA expression in cells from SP-A2/2 sham mice, the difference compared with sham and LPS-treated WT mice was not statistically significant, and the mRNA concentrations did not translate into higher concentrations of protein expression. To determine the functional activity of the iNOS protein, we quantified NOS activity in lung homogenates (Figure 6D). This assay provides a sensitive and specific measurement of NOS activity, because it measures the direct enzymatic conversion of radioactive arginine to citrulline. Because LPS stimulation induces iNOS protein expression and because treatment with the iNOS-specific inhibitor 1400W completely abrogated the attenuated AR in LPS-treated SP-A2/2 mice (Figure 7), the activity measured in this assay is likely iNOS-dependent. As expected, activity was greatest in the LPS-treated groups compared with the sham groups, and the LPS-treated SP-A2/2 mice exhibited the highest levels of activity. The thiol groups constitute other reactive targets for NO. Accumulating evidence indicates that the bioactivity of NO is conveyed largely through the covalent modification of cysteine sulfurs by NO to form SNOs (29–31). SNOs have minimal reactivity with oxygen and superoxide, and thereby limit the formation of reactive nitrogen species such as peroxynitrite (32). SNOs are found in higher concentrations in the lung, and were shown to represent a major source of bronchodilatory NO bioactivity (32). Although not statistically significant, a trend toward greater SNO concentrations was evident in LPS-treated SP-A2/2 mice compared with their WT counterparts (Figure 6E). NOS Inhibition Completely Abrogates the Attenuated AR in SP-A2/2 LPS Mice

Because LPS-treated SP-A2/2 mice demonstrated a relative airway hyporesponsiveness, with elevated nitrite concentrations, iNOS

expression, and NOS activity compared with LPS-treated WT mice, we tested the effect of 1400W, an iNOS-specific inhibitor, on AR in the mice. Physiologic parameters and AR at baseline (Table 1) or with Mch challenge (data shown as sham in Figure 7) did not differ between sham WT and SP-A2/2 mice treated with 1400W or saline vehicle (data not shown). When LPStreated SP-A2/2 mice were pretreated with 1400W, AR to MCh exceeded the levels of AR in the vehicle/LPS-treated WT mice (Figure 7). Thus, the relative airway hyporesponsiveness observed in LPS-treated SP-A2/2 mice was completely abrogated when the mice were treated with 1400W. AR concentrations in 1400W/LPS-treated WT mice also exceeded the AR levels of LPS-treated WT mice. The increase in AR in both the 1400W/ LPS-treated WT and SP-A2/2 mice over the LPS-treated WT mice appears to result primarily from changes in G (Figure 7C), suggesting that the inhibition of iNOS promotes further parenchymal tissue resistance or airflow heterogeneity. BALF cell counts and total protein concentrations were similar among 1400W-treated sham and LPS-treated WT or SP-A2/2 mice (data not shown). Therefore, abrogation of the attenuated AR in LPS-treated SP-A2/2 mice was not attributable to differences in common LPS-associated inflammatory parameters compared with WT mice, but appears to be mediated through an iNOS-dependent mechanism.

DISCUSSION Whereas we and other groups demonstrated a role for SP-A in regulating various inflammatory responses, our new data indicate a novel role for SP-A in altering a very significant physiologic component of many lung diseases, AR, and reveal a possible mechanism involving the regulation of iNOS by SP-A. Our findings show a disconnect between inflammation and AR that is mediated by SP-A. Specifically, SP-A2/2 mice treated with LPS exhibit attenuated Mch-induced AR compared with their WT counterparts, and this attenuation correlated with elevated concentrations of NO derivatives rather than concentrations of


Figure 2. WT and SP-A2/2 mice exhibited similar LPS-associated inflammatory responses. (A) Bronchoalveolar lavage fluid (BALF) cells were counted, and cell differentials were determined using standard hematologic criteria. We derived neutrophil counts/group by multiplying the percent of neutrophils by the total BALF cell counts/group. (B ) BALF supernatant was collected and analyzed for TNF-a and keratinocyte chemoattractant (KC) secreted protein concentrations, using ELISA. Results are reported as picograms per milliliter (pg/ml) of BALF supernatant. Values are means 6 SEM (n 5 10–16 mice/group; *P < 0.05 compared with sham mice, in whom concentrations were not detectable). (C ) BALF supernatant was analyzed for total protein concentrations. Results are reported as micrograms per milliliter (mg/ml) of BALF supernatant. Values are means 6 SEM (n 5 11–18 mice/group; *P < 0.05, compared with sham mice).

inflammatory cytokines or cells. Moreover, treatment with an iNOS-specific inhibitor completely abrogated the relative airway hyporesponsiveness in LPS-exposed SP-A2/2 mice, further supporting a NO-mediated mechanism. To investigate whether differences in LPS-induced inflammation are responsible for the reduced AR observed in SP-A2/2 mice, we analyzed the numbers and types of lung lavage fluid cells, measured lavage concentrations of inflammatory mediators, and histologically assessed the level of influx of inflammatory cells into the lung. Inflammatory cells were recruited to LPS-exposed lungs, but no significant difference was evident in

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Figure 3. LPS exposure resulted in robust neutrophilic infiltration in lungs of WT and SP-A2/2 mice. After either sham or LPS exposure, lungs were fixed with 4% paraformaldehyde at 25 cm H2O pressure and paraffin-embedded. Lung-tissue sections stained with hematoxylin and eosin were evaluated by light microscopy. Large areas of neutrophilic infiltration are evident in the perivascular and peribronchiolar areas of lungs in both LPS-treated WT mice (A and B) and SP-A2/2 mice (C and D). Arrows indicate areas of higher magnification. Scale bars, 50 mm for images at 320 magnification (A and C ), and 25 mm for images at 340 magnification (B and D). Photomicrographs are representative of sections from four mice per group.

the numbers or types of cells recovered in the lung lavage fluid, or observed via histology around the airways, between WT and SP-A2/2 mice. No differences were evident in total protein or cytokine protein concentrations in the lavage. It is not clear why TNF-a protein concentrations were not elevated to a greater degree in LPS-treated SP-A2/2 mice, because we previously found that these mice had elevated concentrations of this cytokine in their BALF (14). However, several differences exist between previous studies and our present study with regard to the time point analyzed (3 hours versus 4 hours), the method for delivery of LPS (intratracheal instillation versus aerosol inhalation), and the strain of mice (J129 versus C57BL/6). Nonetheless, the data show that the diminished AR observed in LPS-exposed SP-A2/2 mice is not attributable to differences in lung inflammatory indices relative to WT mice.

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Figure 4. Surfactant protein–D (SP-D) concentrations increased similarly in WT and SP-A2/2 mice. BALF supernatant was collected and analyzed for SP-D concentrations by Western blotting. (A) Representative blot of four independent experiments. (B) Each band was quantified as described in MATERIALS AND METHODS, and compared with the positive control on each blot (i.e., 15 ng of recombinant SP-D). SP-D expression values are expressed as percent expression relative to positive control. Values are means 6 SEM (n 5 6–10 mice/group; *P < 0.05, compared with sham mice).

The expression of another surfactant-associated protein and innate immune molecule, SP-D, is upregulated during allergeninduced inflammation in mice (26, 33). SP-D was shown to regulate the development of AR and airway inflammation

negatively, in part by modulating the function of alveolar macrophages (26) or by inhibiting the synthesis of cytokines (33). Therefore, we measured concentrations of SP-D protein in the BALF, and found that SP-D concentrations were similarly elevated in SP-A2/2 and WT mice exposed to LPS, indicating that the reduced AR cannot be attributed to differences in BALF SP-D concentrations. Mechanisms involved in airway hyperresponsivness can include an increase in smooth muscle contraction and impaired smooth muscle relaxation (21, 27, 28). Therefore, we compared the contractile and relaxation properties of isolated bronchial rings from WT and SP-A2/2 mice. We found no differences between groups in terms of the receptor-independent contraction elicited by KCl, the contraction in response to the muscarinic receptor agonist carbachol, or the relaxation evoked by isoproterenol. These results indicate that an underlying physiologic defect in ASM properties is not responsible for the reduced AR observed in SP-A2/2 mice. Our results, instead, point to a NO-mediated mechanism for the attenuated AR. For instance, the amount of nitrite, a byproduct of NO production, was higher in BALF from the lungs of SP-A2/2 than of WT mice exposed to LPS. These results are consistent with those from a study by LeVine and colleagues (5) demonstrated that concentrations of nitrite were greater in SPA2/2 mice than in WT mice after intratracheal infection with P. aeruginosa, and with our previous observations that SP-A2/2 mice had higher concentrations of nitrite in their lung lavage fluid than did WT mice after intratracheal LPS challenge (14). In addition, we found that expression levels of iNOS and NOS activity, presumably of the iNOS isoform, were significantly elevated in LPS-treated SP-A2/2 compared with WT mice.

Figure 5. Airway smooth muscle (ASM) contractile and relaxation responses were similar between WT and SP-A2/2 mice. Bronchial rings were excised from untreated WT and SP-A2/2 mice, and suspended in a Krebs’ buffer bath. (A) After KCl washout, increasing doses of carbachol were added, and force was measured. Percent contraction relative to maximum contraction was calculated for each ring. Inset: Maximum contraction did not differ between the two groups. (B) After the final dose of carbachol, increasing doses of isoproterenol were added, and force was measured again. Percent relaxation relative to maximum contraction was calculated for each ring. Values are means 6 SEM (n 5 6 mice/ group).


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Figure 6. Nitric oxide (NO) derivatives were elevated in LPS-treated SP-A2/2 mice. Single-cell suspensions from lung-tissue digests were subjected to flow cytometric analysis. (A) Nitrite concentrations. Results are reported as mean micromolar (mM) concentration in BALF 6 SEM (n 5 8–10 mice/group; *P < 0.05, compared with shamtreated WT mice; ^P < 0.05, compared with shamtreated SP-A2/2 mice and LPS-treated WT mice). (B) Inducible nitric oxide synthase (iNOS) mRNA. Results reported as fold difference in iNOS/cyclophilin values, compared with WT sham control mice (3–5 mice/group in three independent experiments; *P < 0.05, compared with sham-treated mice; ^P < 0.05, compared with LPStreated WT mice). (C ) Total percent of lung-digest cells expressing iNOS (two mice/group in four independent experiments; *P , 0.05, compared with sham mice; ^P < 0.05, compared with LPS-treated WT mice). (D) NOS activity. Results are reported as fold difference in average activity/mg protein compared with WT sham control mice (3–5 mice/group in three independent experiments; *P < 0.05, compared with sham-treated mice; ^P < 0.05, compared with LPS-treated WT mice). (E ) S-nitrosothiol (SNO) concentrations. Results are reported as mean pmol of SNO/mg of protein 6 SEM (3–4 mice/group in three independent experiments; *P < 0.05, compared with sham-treated mice; ^P < 0.05, compared with LPS-treated WT mice).

NO is produced in many cell types and regulates several vital functions, such as vasodilation, bronchodilation, and microbicidal activation. Diseases of airway inflammation, such as asthma, were associated with increased concentrations of NO and its metabolites in expired breath and expired breath condensates (34, 35), but whether or not NO is a proinflammatory or anti-inflammatory mediator remains unresolved. Although the relationship of NO to AR is also controversial, endogenous NO was shown to protect against bronchoconstrictor stimuli (36, 37). Studies using mice genetically deficient in various NOS isoforms, or mice subjected to pharmacologic pan-NOS inhibition, showed that airway inflammation is unrelated, positively related, or negatively related to the production of NO, and that NO is inversely correlated with, or unrelated to, AR (38–41). These conflicting results are likely attributable to differences in methodology, including the method of inducing lung inflammation, and the method of measuring AR. Because our data show that LPS-challenged SP-A2/2 mice have elevated NO-associated indices and attenuated AR compared with LPSchallenged WT mice, we tested the hypothesis that the enhanced production of NO contributes to the diminished AR observed in SP-A2/2 mice by pretreating mice with 1400W, an iNOS-specific inhibitor. The treatment of SP-A2/2 mice with 1400W reversed the attenuation in AR observed in response to LPS exposure. These results are consistent with our hypothesis that the elevated expression of iNOS observed in LPS-treated SP-A2/2 mice contributes to the elevated concentrations of NO that diminish AR.

Although elevated concentrations of NO may be advantageous in the short term to preserve airway patency in the midst of an acute inflammatory response, increased sustained NO production over time was associated with adverse airwayremodeling affects (42). Baseline BALF nitrite was elevated in sham-treated SP-A2/2 mice and not in sham-treated WT mice. Nonetheless, BALF cell counts, lung structure, and respiratory system physiologic measurements were similar among these mice, arguing against a deleterious effect of increased NO production during this time period. Moreover, the elevated nitrite concentrations may have resulted from higher concentrations of the constitutive NOS isoforms at baseline, but in the context of LPS-induced inflammation, the iNOS isoform is preferentially upregulated. NO is produced by NOS, and can react with glutathione in the lungs to form S-nitrosoglutathione (GSNO), the most common SNO in the airway. This reactive molecule may directly dilate bronchial smooth muscle, cause protein S-nitrosylation (and thereby affect signaling molecules, enzymes, and transcription factors), or be degraded to oxidized glutathione and ammonias by the enzyme S-nitrosoglutathione reductase (GSNOR). In concordance with the responses observed in LPS-treated SP-A2/2 mice in our study, Que and colleagues recently showed that ovalbumin-treated GSNOR2/2 mice have attenuated AR, despite exhibiting robust lung inflammation (21). Here, we show a trend toward greater SNO concentrations in LPS-treated SP-A2/2 mice compared with their WT counterparts. Thus, the balance between the synthesis and metabolism of GSNO appears to be

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Figure 7. Treatment with the iNOSspecific inhibitor 1400W reversed relative airway hyporesponsiveness in LPS-treated SP-A2/2 mice. Mice were treated with or without 1400W, and were challenged with saline or LPS. Respiratory-system physiologic measurements, including total respiratory system resistance (RT), central airway resistance (RN), tissue damping (G), and tissue elastance (H ) and compliance (C ), were acquired after aerosolized MCh challenge (0 mg/ml, 10 mg/ml, 25 mg/ml, and 100 mg/ml). (A) 1400W treatment enhanced RT in LPS-treated WT mice (open circles), and abrogated the attenuated airway responsiveness in LPStreated SP-A2/2 mice (open squares). (B ) 1400W treatment enhanced RN in LPStreated SP-A2/2 mice (open squares). (C ) 1400W treatment enhanced G in LPStreated WT mice (open circles) and LPStreated SP-A2/2 mice (open squares). (D) 1400W treatment enhanced H in LPStreated SP-A2/2 mice (open squares). The average of each physiologic measurement per Mch dose was graphed as fold change from baseline measurement (n 5 6–12 mice/group; *P < 0.05, comparing LPStreated mice with sham-treated mice; ^P < 0.05, comparing 1400W/LPS–treated SP-A2/2 mice with vehicle/LPS–treated SP-A2/2 mice and vehicle/LPS–treated WT mice).

important in LPS-induced AR, and this balance represents a future area of investigation in determining the mechanisms involved in the opposing phenotypic differences in AR between WT and SP-A2/2 mice.

The negative regulation by SP-A of the LPS-stimulated production of NO, and in consequence, the enhanced AR may be of potential clinical significance, because recent studies reported associations between surfactant protein polymorphisms and a


variety of lung diseases (43–48). In addition, the importance of NO as a marker for inflammation in human airway diseases was recently reported. Although a deficiency of surfactant proteins in mice is clearly related to an increased susceptibility to infectious lung diseases, our study raises the intriguing possibility that deficiencies of surfactant, perhaps via surfactant protein polymorphisms or inactivation, may modify the susceptibility of the host in terms of other airway diseases. This study demonstrates that the presence of SP-A increases the susceptibility to LPS-induced elevated AR, and highlights the importance of assessing the contributions of surfactant proteins in human inflammatory lung disease. Author Disclosure: A.M.P. has received sponsored grants from the National Institutes of Health (more than $100,001). J.K.L.W. holds stock ownership/ options for Johnson & Johnson (less than $5,000), General Electric (less than $1,000), Kimberly Clark (less than $5,000), and 3M (less than $5,000). L.A.M. holds stock ownership/options with Pfizer (less than $1,000) and General Electric (less than $1,000), and is an employee of the National Institutes of Health. C.G. received compensation for serving as an expert witness with Wallace and Graham ($10,001–$50,000), Brayton and Purcell ($5,001–$10,000), Weitz and Luxemberg ($50,001–$100,000), and Waters and Kraus ($10,001–$50,000), has a patent pending with Medimmune for TLR4 hyporesponsive polymorphisms used in respiratory syncytial virus vaccine research (less than $10,000), and is an employee at the National Institutes of Health and National Jewish Health. B.H. has received sponsored grants from the National Institutes of Health (more than $100,001). M.E.S. has received lecture fees from Ono Pharmaceutics. T.R.K. is an employee at the York Technical Institute (YTI). The spouse of D.A.S. has received industry-sponsored grants from Amgen (less than $1,000), Eli Lily (less than $5,000), Novartis (less than $5,000), Roche (less than $1,000), and Auxilium (less than $1,000), and has a patent pending with Duke University (Reference Number 2809 for Use of Imipramine to Treat Asthma). T.J.M. recently filed two new patents via Duke University, which involve the use of gastrin-releasing peptide blockade to treat asthma or any inflammatory condition, and she has not yet communicated with any commercial entity regarding these pending patents. She also received sponsored grants from the National Institutes of Health (more than $100,000) and the American Asthma Foundation (more than $100,000). M.E.S. also has a patent pending with the Polycystic Kidney Disease Foundation (pending application from a previous grant sponsor) and from University of California Santa Barbara (pending application from a previous research laboratory). L.Q. has received funding from the National Institutes of Health (more than $100,000). J.R.W. holds the license to patent in which he is a co-inventor from N30 Pharma, LLC, and has received sponsored grants from the National Institutes of Health (more than $100,000) and the Veterans Administration (more than $100,000). None of the other authors have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. Acknowledgments: The Duke Human Vaccine Institute Flow Cytometry Core Facility is supported by National Institutes of Health grant AI-051445. The LPS aerosol exposures and airway physiologic measurements were performed in the Inhalational Toxicology Core of the Duke Center for Comparative Biology of Vulnerable Populations, which is supported by National Institutes of Health grant 5P30ES011961.

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