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Final Report: SP-A and SP-D in Environmental Lung Disease
EPA Grant Number: R825702C001Subproject: this is subproject number 001 , established and managed by the Center Director under grant R825702
(EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).
Center: Environmental Lung Disease Center (National Jewish Medical and Research Center)
Center Director: Mason, Robert
Title: SP-A and SP-D in Environmental Lung Disease
Investigators: Mason, Robert P.
Institution: National Jewish Medical and Research Center
EPA Project Officer: Glenn, Barbara
Project Period: February 16, 1998 through February 28, 2003 (Extended to February 28, 2004)
RFA: Environmental Lung Disease Center (National Jewish Medical and Research Center) (1998)
Research Category: Targeted Research
Description:
Objective:The objectives of this research project were to determine: (1) the binding of pulmonary surfactant protein (SP) D to carbohydrates and to evaluate serum and lavage SP-D as a biomarker for ozone and particulates; (2) if ozone stimulates the secretion of inflammatory chemokines by alveolar type II cells, type I-like cells, and pulmonary macrophages in vitro; (3) how ozone exposure alters lipogenesis in type II cells; and (4) if ozone stimulates the expression and secretion of inflammatory chemokines and SP-A and SP-D in type II cells in vivo.
Summary/Accomplishments (Outputs/Outcomes):Studies on the Binding of SP-D to Yeast and Carbohydrates
The first study was on the binding of SP-A and SP-D, members of the collectin family of calcium-dependent lectins, to Saccharomyces cerevisiae and Aspergillus fumigatus. SP-D, but not SP-A, bound to yeast walls in a calcium-dependent manner. Studies with β(1→3)-gluconase and inhibition by pustulan indicated that the binding was through β(1→6) glycan. This study concentrated on determining the specificity for SP-D for certain glycosidic bonds.
The next study was done by computer modeling to demonstrate binding by both vicinal hydroxyls at positions 3,4 and 2,3 could serve as sites for carbohydrate recognition. The binding to the vicinal 2,3 position was unexpected and demonstrated that SP-D could bind to internal sugars and not just end-terminal sugars.
The final paper in this series of experiments demonstrated the critical roles for Arg 343 in carbohydrate binding. Dr. Allen used both computer modeling as well as site-directed mutagenesis and recombinant proteins to demonstrate the critical role of this amino acid in determining carbohydrate specificity for binding of SP-D to its ligands.
Maintenance of the Type I and Type II Cell Phenotypes In Vitro
We have developed a culture system for maintaining rat type II cells in a differentiated state as a monolayer so that exposures can be done and secretion or apical uptake can be measured. In brief, rat type II cells are plated into the upper chamber of a Millicell, which is coated with a mixture of Matrigel and collagen and cultured with 5 percent rat serum (RS) and 10 ng/mL keratinocyte growth factor (KGF). For maximal differentiation, we add 108 M dexamethasone for the last 4 days. These cells express the surfactant proteins at high levels and secrete phosphatidylcholine and SP-A and SP-D into the apical fluid. Defining culture conditions for type I cells has been more problematic. Instead of isolating type I cells, which is extremely hard and difficult to maintain in vitro, we have chosen to isolate type II cells and have them transdifferentiate into type I-like cells in vitro. This also is the approach taken by other investigators. These type I-like cells can be derived from type II cells by growing them with 5 percent fetal bovine serum (FBS) in the absence of other differentiation factors. Our outcome variables for defining type I-like cells are appearance: histology; tomato lectin staining; T1α and caveolin protein expression by Western analyses and immunostaining; and T1α, aquaporin V, and caveolin mRNA levels. There always will be a concern that these might not be fully differentiated type I cells or may not fully reproduce the properties of type I cells in the lung in vivo, but this is the current state of alveolar epithelial cell culture. One other option was to use a mouse cell line (E-10), which expresses type I cell markers. We have obtained this cell line from Dr. Malkinson and cleared it from mycoplasma infection. Ozone and the oxidized cholesterol products can damage this cell line, but it does not respond to KGF in our preliminary studies.
Cytokines and Chemokines Expressed by Type I and Type II Cells
We have found that chemokine secretion (MCP-1, MIP-2) and mRNA levels (CINC-1, CINC-2α, and CINC-3) are not dependent upon the state of type II cell differentiation or SP-A or SP-D secretion. These chemokines are secreted by both type II cells and type I-like cells in vitro. These chemokines are regulated by cytokines such as the mixture of IL-1β, TNF α, and IFN-γ, or IL-1β above. CINC-2β mRNA levels, however, are very dependent on the state of differentiation of type II cells. CINC-2β mRNA levels are expressed only in the type II cell phenotype and are not stimulated by cytokines. This observation was made with quantitative real-time polymerase chain reaction (RT-PCR) and confirmed by Northern analyses. There is one gene for CINC-2, and the expression of CINC-2α and CINC-2β is regulated by differential RNA splicing. Because of the minor changes in protein structure, we are not confident in the protein measurements of CINC-2β determined by enzyme-linked immunosorbent assay. By in situ hybridization CINC-2β is expressed only in type II cells and Clara cells in the rat lung. Another type II restricted chemokine is LIX (CXCL5, ENA-78). On the other hand, MCP-1 (CCL-2) is expressed at a higher level by the type I-like cell phenotype. Both phenotypes secrete chemokines in response to LPS.
Effects of Ozone on Alveolar Epithelial Cells In Vitro
We have done several studies on exposures of these monolayers to oxidants and ozone. Both type II cells and type I-like cells are sensitive to relatively low levels of ozone (0.1 ppm for 60 minutes). Type I-like cells are more sensitive to ozone than are differentiated type II cells. Our initial studies have shown that there was very little chemokine secretion in response to ozone but that these cultured epithelial cells could secrete chemokines in response to IL-1β or endotoxin. We then tested if IL-1β could prime the cells to produce chemokines in response to ozone or if ozone would sensitize the cells to IL-1β. These studies with ozone or IL-1β priming were negative. IL-1β, however, stimulated cytokine secretion after ozone exposure, so their failure to secrete chemokines was not caused by the toxicity of ozone. These studies were repeated with endotoxin as a priming agent and again we did not find a synergistic interaction with endotoxin and ozone. There was no consistent priming effect that could be demonstrated in vitro. These observations are different from some reports in vivo, where pre-exposure to endotoxin enhances the chemokine and inflammatory response to a subsequent ozone exposure.
Gene Profiling of the Effect of Ozone on Type II Cells and Type I-like Cells In Vitro
To understand the molecular basis of oxidative stress in alveolar epithelial cells, primary cultures of differentiated and dedifferentiated (type I-like phenotype) alveolar type II cells were exposed to 0.1 ppm ozone or air for 1 hour. The dedifferentiated, type I-like phenotype was more sensitive to ozone than the type II cell phenotype. The differentiated type II cells were cultured in transwells with KGF, rat serum, and dexamethasone, and the dedifferentiated alveolar epithelial cells were grown with fetal bovine serum and dexamethasone. The mRNA from both phenotypes was collected at 4 hours and 24 hours postexposure to air or ozone and used for gene expression profiling using Affymetrix microarrays. Four independent experiments were done under all conditions. RT-PCR and Western blot analysis are being used to verify significant changes in mRNA and protein levels of some early response and stress genes. The genomic pattern for alveolar epithelial cells in response to ozone involves stress responses, chemokine production, transcription factors, cell proliferation, water channels, and lipid metabolism. The air-exposed cultures also allow for comparison of differentiated and dedifferentiated alveolar epithelial cells. The genome wide mRNA survey provided insight into signal pathways activated by oxidant stress and how cellular protection mechanisms are initiated. In addition, many of the changes in mRNA levels for stress genes were confirmed at the protein level by immunoblotting.
Effects of Ozone on Alveolar Epithelial Cells In Vivo
Our proposed studies are to expose animals to ozone in vivo and then to isolate macrophages and type II cells. These studies are on hold until we identify a more robust signal in response to ozone in vitro.
Previously, we proposed that serum SP-D would be a useful serum marker of lung inflammation. We recently have completed a study with Dr. Renaud Vincent on the effects of ozone and concentrated ambient air particles on serum and lavage levels of SP-D in mice. Ozone but not particles increased serum and lavage SP-D. The effect was restricted to ozone, and even high doses of concentrated particles produced very little effect on serum SP-D levels. In other studies, serum levels of SP-D increased with antigen instillation into sensitized mice and after instillation of bleomycin.
Significance
Importance to the Scientific Community. The most interesting finding is that epithelial cells (and macrophages) do not secrete chemokines in direct response to ozone. Presumably the signal for chemokine production is from ozone-injured cells communicating with other noninjured cells to produce chemokines. We have preliminary data that ozone-injured cells secrete IL-1α, which in turn stimulates other cells to secrete chemokines. We showed that both type I can type II cells can secrete chemokines. There are two chemokines, however, that are expressed preferentially by type II cells. These are CINCX-2β and LIX. The second important finding is that type II cells are much more resistant to ozone than type I cells. There are several possibilities for this resistance, but one is that type II cells express more glutaredoxin and the collectins SP-A and SP-D. A third finding is that measuring serum SP-D in mice can assess ozone damage to the lung. To our knowledge, this is the first serum measurement that correlates well with lung injury. Interestingly, particulate exposure does not increase serum SP-D in mice. We also have evaluated SP-D as in important component of innate immunity. We have shown that the collectin SP-D binds to internal sugars in a polysaccharide chain as well as the terminal system. SP-D binds to vinyl hydroxyls in certain sugars in a specific configuration.
Importance to the U.S. Environmental Protection Agency. The studies on gene expression in type I and type II cells point to the role of glutaredoxin, heat shock protein 70, hemoxygenase 1, metallothionein, SP-A, and SP-D as important protective proteins in the response to ozone. Theoretically, these could be therapeutic targets to prevent injury caused by oxidant gases. The other important finding is that in rodents, serum SP-D could be used as a biomarker for ozone exposure. This is the first serum marker for the effect of ozone to our knowledge.
Journal Articles on this Report: 10 Displayed | Download in RIS Format
| Other subproject views: | All 12 publications | 10 publications in selected types | All 10 journal articles |
| Other center views: | All 69 publications | 39 publications in selected types | All 39 journal articles |
| Type | Citation | ||
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Allen MJ, Harbeck R, Smith B, Voelker DR, Mason RJ. Binding of rat and human surfactant proteins A and D to Aspergillus fumigatus conidia. Infection and Immunity 1999;67(9):4563-4569. |
R825702 (Final) R825702C001 (Final) |
not available |
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Allen MJ, Voelker DR, Mason RJ. Interactions of surfactant proteins A and D with Saccharomyces cerevisiae and Aspergillus fumigatus. Infection and Immunity 2001;69(4):2037-2044. |
R825702 (Final) R825702C001 (Final) |
not available |
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Allen MJ, Laederach A, Reilly PJ, Mason RJ. Polysaccharide recognition by surfactant protein D: novel interactions of a C-type lectin with nonterminal glucosyl residues. Biochemistry 2001;40(26):7789-7798. |
R825702 (Final) R825702C001 (Final) |
not available |
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Allen MJ, Laederach A, Reilly PJ, Mason RJ, Voelker DR. Arg343 in human surfactant protein D governs discrimination between glucose and N-acetylglucosamine ligands. Glycobiology 2004;14(8):693-700. |
R825702 (Final) R825702C001 (Final) |
not available |
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Kanehiro A, Lahn M, Makela MJ, Dakhama A, Fujita M, Joetham A, Mason RJ, Born W, Gelfand EW. Tumor necrosis factor-alpha negatively regulates airway hyperresponsiveness through gamma-delta T cells. American Journal of Respiratory and Critical Care Medicine 2001;164(12):2229-2238. |
R825702 (Final) R825702C001 (Final) |
not available |
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Kumarathasan P, Blais E, Goegan P, Yagminas A, Guenette J, Adamson IY, Crapo JD, Mason RJ, Vincent R. 90-day repeated inhalation exposure of surfactant protein-C/tumor necrosis factor-alpha, (SP-C/TNF-alpha) transgenic mice to air pollutants. International Journal of Toxicology 2005;24(1):59-67. |
R825702 (Final) R825702C001 (Final) |
not available |
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Manzer R, Wang J, Nishina K, McConville G, Mason RJ. Alveolar epithelial cells secrete chemokines in response to IL-1beta and lipopolysaccharide but not to ozone. American Journal of Respiratory Cell and Molecular Biology 2006;34(2):158-166. |
R825702 (Final) R825702C001 (Final) |
not available |
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Pan T, Nielsen LD, Allen MJ, Shannon KM, Shannon JM, Selman M, Mason RJ. Serum SP-D is a marker of lung injury in rats. American Journal of Physiology-Lung Cellular and Molecular Physiology 2002;282(4):L824-L832. |
R825702 (Final) R825702C001 (Final) |
not available |
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Taylor MD, Van Dyke K, Bowman LL, Miles PR, Hubbs AF, Mason RJ, Shannon K, Reasor MJ. A characterization of amiodarone-induced pulmonary toxicity in F344 rats and identification of surfactant protein-D as a potential biomarker for the development of the toxicity. Toxicology and Applied Pharmacology 2000;167(3):182-190. |
R825702 (Final) R825702C001 (Final) |
not available |
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Zhang F, Pao W, Umphress SM, Jakowlew SB, Meyer AM, Dwyer-Nield LD, Nielsen LD, Takeda K, Gelfand EW, Fisher JH, Zhang L, Malkinson AM, Mason RJ. Serum levels of surfactant protein D are increased in mice with lung tumors. Cancer Research 2003;63(18):5889-5894. |
R825702 (Final) R825702C001 (Final) |
not available |
air toxics, acute lung injury, air pollutants, airway disease, animal studies, environmental toxicant, exposure, genetic susceptibility, health effects, human exposure, human health risk, lung disease, lung epithelial cells, occupational disease, occupational exposure,
,
Air, Scientific Discipline, Health, RFA, PHYSICAL ASPECTS, Risk Assessments, Disease & Cumulative Effects, Health Risk Assessment, Physical Processes, Atmospheric Sciences, particulate matter, Environmental Chemistry, tropospheric ozone, aerosols, health effects, respiratory problems, ambient air, ozone, environmental health effects, Aspergillus spores, harmful environmental agents, human health risk, lung inflammation, cytokine production, ambient particulates, air pollutants, air toxics, chronic health effects, human health effects, particulates, respiratory, air pollution, airway disease, atmospheric chemistry, lung, lung disease, epithelial cells, exposure, pulmonary, airway epithelial cells, surfactant protiens, human exposure, PM, pulmonary disease, particulate exposure
Relevant Websites:
http://www.nationaljewish.org/patient-info/progs/med/environmental/index.aspx 
Progress and Final Reports:
Original Abstract
Main Center Abstract and Reports:
R825702 Environmental Lung Disease Center (National Jewish Medical and Research Center)
Subprojects under this Center:
(EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).
R825702C001 SP-A and SP-D in Environmental Lung Disease
R825702C003 Adaptation to Nitrogen Dioxide: Role of Altered Glycolytic Pathway Enzyme Expression and NF-κB-Dependent Cellular Defenses Against Apoptosis
R825702C005 Inhalation of Particulate Matter Alters the Allergic Airway Response to Inhaled Allergen
R825702C006 Particle-Induced Lung Inflammation and Extracellular EC-SOD
R825702C007 Indoor-Outdoor Relationships of Airborne Particle Count and Endotoxin Concentrations
R825702C008 The Role of Mitochondrial DNA Mutations in Oxidant-Mediated Lung Injury
R825702C009 Immunopathogenesis of Hypersensitivity Pneumonitis in the Mouse
R825702C010 Activation of Natural T Lymphocytes by Diesel Exhaust Particulates Leads to Their Production of Interleukin-4 and TH2 Lymphocyte Differentiation to Allergen
R825702C011 Latex Antigen Levels During Powdered and Powderless Glove Use
R825702C012 Adjuvant Effects of Ozone in a Model of Allergen-Induced Airway Inflammation and Hyperresponsiveness
R825702C013 Acute Exposure to Particulate Air Pollution in Childhood Asthma
R825702C014 Mechanisms of Ozone Toxicity to the Lung