| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
|
FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online June 18, 2004 as doi:10.1096/fj.04-1518fje. |
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
,1
* Division of Pulmonary and Critical Care Medicine,
Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts, USA; and
Thermal and Mountain Medicine Division, U.S. Army Research Institute of Environmental Medicine, Natick, Massachusetts, USA
1 Correspondence: Pulmonary and Critical Care Division, Brigham and Women’s Hospital, 75 Francis St., Boston, MA 02115, USA. E-mail: mperrella{at}rics.bwh.harvard.edu
SPECIFIC AIM
Our aim was to elucidate the role of epithelial cell nitric oxide synthase-2 (NOS2) expression in the development of physiologic lung dysfunction, surfactant dysfunction, and loss of surfactant protein-B expression during acute lung injury.
PRINCIPAL FINDINGS
1. Nebulized LPS results in severe physiologic lung dysfunction in wild-type (WT) but not in NOS2–/– mice
Lung physiology at baseline (vehicle) and after two exposures to nebulized LPS was measured in WT and NOS2–/– mice (Fig. 1
). Before LPS treatment, tissue resistance (G), and lung elastance (H, a measure of lung stiffness and inverse of compliance) were nearly identical in both groups. Two exposures of LPS 24 h apart caused a significant increase in H (1.7±0.20-fold, P<0.05) and G (2.0±0.16-fold, P<0.05) in WT mice. In NOS2–/– mice, lung elastance and tissue resistance did not change significantly from baseline after LPS exposure.
|
2. Surfactant function is abnormal in LPS-treated WT mice but remains normal in LPS-treated NOS2–/– animals
Dynamic surface tension vs. surface area profiles were measured in surfactant samples isolated from WT and NOS2–/– mice. Before LPS exposure, surfactant function was normal and nearly identical in both groups of animals. In LPS-treated WT mice, surfactant surface tension-to-surface area ratio increased (representative of significant dysfunction). Surfactant function in NOS2–/– mice was minimally affected by LPS exposure with a surface tension-to-surface area ratio similar to that of untreated animals.
3. LPS treatment induces a NOS2-specific reduction of SP-B
We previously demonstrated that LPS-treated WT animals exhibited decreased SP-B lung content vs. control animals whereas levels of SP-A and SP-C were not affected by LPS treatment. Therefore, we assessed SP-B mRNA levels in lung tissue of WT and NOS2–/– animals (Fig. 2
A). In WT mice, SP-B mRNA decreased 87% after LPS exposure whereas in NOS2–/– mice, LPS exposure had no significant effect on SP-B RNA levels. Lung immunostaining was performed for NOS2, SP-B, and CD45 (leukocyte common antigen) in WT and NOS2–/– animals (Fig. 2B
). NOS2 staining was absent in both groups of vehicle-treated animals (a, b). NOS2 staining was present in parenchymal and inflammatory cells in LPS-treated WT mice (c) but absent in LPS-treated NOS2–/– mice (d). SP-B staining was present in alveolar epithelial cells (arrows) in vehicle-treated WT and NOS2–/– animals (e, f). SP-B content was significantly reduced in LPS-treated WT animals (g). In contrast, LPS-treated NOS2–/– animals retained SP-B expression (h) unchanged from vehicle-treated animals. LPS-treated WT and NOS2–/– animals showed equivalent staining for inflammatory cells (CD45, i, j).
|
4. BAL cell counts and cytokine levels do not differ between LPS-treated NOS2–/– and WT animals
Cell counts, cell count differentials, and cytokine levels were measured in bronchoalveolar lavage (BAL) fluid. At baseline, WT and NOS2–/– mice exhibited similar low-level cell counts and differentials, with the macrophage as predominant cell type. After 48 h of LPS exposure, a significant and similar increase in the number of inflammatory cells, predominantly neutrophils, was observed in both groups of animals (WT: 1.3x106±0.16 cells, 82% neutrophils; NOS2–/–: 1.3x106±0.23 cells, 87% neutrophils P=n.s. for WT vs. NOS2–/–). Interleukin (IL) -6 and tumor necrosis factor (TNF-
) levels were undetectable in control (vehicle-treated) animals and significantly elevated to a similar degree in WT and NOS2–/– animals after 48 h of LPS treatment.
5. NOS2 expression in lung parenchymal cells is critical for development of LPS-induced acute lung injury
To determine whether NOS2 expression in inflammatory cells or parenchymal cells was critical for development of LPS-induced acute lung injury, bone marrow transplantation (BMT) was performed to derive chimeric animals for NOS2 (NOS2–/– cells into WT recipients [NOS2–/–
WT]; WT cells into NOS2–/– recipients [WTNOS2–/–]).
Lung physiology at baseline (vehicle) and after two exposures to nebulized LPS was measured in chimeric mice. Before LPS treatment (vehicle group), G and H were normal and nearly identical in both groups of animals. In NOS2–/–WT mice, LPS exposure caused an increase in G (1.5±0.08-fold) and a significant increase in H (2.1±0.10-fold, P<0.05) compared with vehicle-treated animals. G and H did not change significantly after LPS exposure in WTNOS2–/– mice. Thus, chimeric animals exhibited indices of acute lung injury mirroring the recipient genotype. Lung immunostaining was performed for SP-B in chimeric animals. As in nontransplanted animals, SP-B staining was present in alveolar epithelial cells in both groups of vehicle-treated animals. SP-B content was significantly reduced in LPS-treated NOS2–/–WT animals. WTNOS2–/– animals retained SP-B expression, which was unchanged from vehicle-treated animals. Thus, chimeric animals exhibited SP-B lung immunostaining mirroring that of the recipient genotype. Similar to nontransplanted WT and NOS2–/– animals, BAL total cell counts and BAL cytokine levels were not significantly different in the two groups of LPS-treated chimeric animals.
6. Nitric oxide (NO) down-regulates SP-B message and promoter activity in lung epithelial cells
We next determined whether NO, independent of inflammatory cytokines, decreases SP-B expression in lung epithelial cells in vitro. Northern blot analysis was performed on total RNA extracted from mouse lung epithelial (MLE-15) cells treated with NO donor DETA NONOate (3 mM) or vehicle. SP-B mRNA decreased 19%, 31%, and 45% after NO exposure for 6, 12, and 24 h, respectively, compared with vehicle (P<0.05 for 12 and 24 h). Transient transfections were performed using an SP-B promoter/reporter construct (p-1797/+42/LUC) in mouse lung epithelial cells treated with DETA NONOate (0.5 mM, 1 mM) or vehicle. SP-B promoter activity decreased by 48% and 62% with addition of 0.5 mM and 1 mM of DETA NONOate, respectively (P<0.05 for each dose of DETA NONOate).
7. SP-B, but not SP-A nor SP-C, restores normal function to WT surfactant after LPS exposure
Reconstitution studies using SP-A, SP-B, and SP-C were performed to confirm the relationship between surfactant dysfunction and reductions in SP-B levels seen in LPS-treated WT animals. Surfactant samples from two LPS-treated WT mice were pooled, and surfactant function was analyzed at baseline and after addition of physiological equivalents of SP-A, SP-B, or SP-C. Addition of SP-B restored function to abnormal surfactant isolated from LPS-treated WT mice. Addition of SP-A or SP-C did not fully restore normal surfactant function.
CONCLUSIONS AND SIGNIFICANCE
Acute respiratory distress syndrome (ARDS) is a life-threatening clinical condition affecting 150,000 patients a year. ARDS is initiated by an inflammatory injury to the lung that leads to diffuse pulmonary infiltrates, alveolar collapse, and refractory hypoxemia. These changes correlate with loss of lung surfactant, the lipoprotein mixture synthesized and secreted by lung epithelial cells. SP-B is the most critical surfactant protein for maintenance of normal surface film function. NO has been recognized as an essential modulator of lung biology and an important mediator of acute inflammation. In the present study, we demonstrate that epithelial cell expression of NOS2 during severe acute lung injury results in physiologic lung dysfunction (Fig. 1)
, surfactant dysfunction, and transcriptional down-regulation of SP-B expression (Fig. 2)
.
A key observation in our study is that the extent of lung inflammation and production of inflammatory cytokines within BAL fluid was not different between WT and NOS2–/– animals subjected to 48 h of nebulized LPS. Our model reflects severe lung injury once inflammatory cell recruitment has occurred and overwhelming lung injury has developed, mirroring the clinical presentation of patients with ARDS. Our findings complement and add to those of others who found that inflammatory cell expression of NOS2 was critical for development of microvascular protein leak during the first 8 h after sepsis. Our data support the concept that NOS2 expression is critical in different cell types at different times depending on the phase or extent of lung injury.
These findings support a novel role for NOS2 in the pathobiology of acute lung injury. It has been thought that NO generated by activated macrophages in response to bacterial stimulation is released into the extracellular environment, where it can cause surfactant dysfunction by reacting directly with lipid and protein components via generation of peroxynitrite or hydroxyl radical. Our findings suggest that extracellular free radical-mediated changes in surfactant composition may not be primarily responsible for modulating lung dysfunction in ARDS. NOS2 production of NO may be an important regulator of surfactant expression within the alveolar type II epithelial cell by modulating the level of SP-B gene transcription.
These data demonstrate that the NOS2 isoform of NO synthase plays a critical role in regulating expression of SP-B, a protein essential for normal surfactant and lung function. These results indicate that NOS2 expressed in lung epithelial cells decreases expression of SP-B during severe acute lung injury (Fig. 3
). Our findings suggest that therapies for ARDS directed toward modulation of parenchymal cell NOS2 and SP-B expression have potential to ameliorate the disease process, even after development of significant cellular inflammation.
|
FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-1518fje; doi: 10.1096/fj.04-1518fje
This article has been cited by other articles:
|
|
 |
|
  A. V. Fedulov, A. Leme, Z. Yang, M. Dahl, R. Lim, T. J. Mariani, and L. Kobzik Pulmonary Exposure to Particles during Pregnancy Causes Increased Neonatal Asthma Susceptibility Am. J. Respir. Cell Mol. Biol., January 1, 2008; 38(1): 57 - 67. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. M. St. Croix, K. Leelavaninchkul, S. C. Watkins, V. E. Kagan, and B. R. Pitt Nitric Oxide and Zinc Homeostasis in Acute Lung Injury Proceedings of the ATS, October 1, 2005; 2(3): 236 - 242. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
) and tissue elastic (H, 
) moduli. G is proportional to tissue resistance and H to lung elastance (lung stiffness). At baseline (vehicle), both groups had similar values of G and H. In WT mice, LPS exposure caused significant increases in G and H whereas G and H did not change significantly after LPS exposure in NOS2–/– mice. Error bars = SE. (*P<0.05 compared with vehicle-treated WT mice for G and H; **P<0.05 compared with LPS-treated WT mice).
