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1.  A Common Polymorphism in EC-SOD Affects Cardiopulmonary Disease Risk by Altering Protein Distribution 
The enzyme extracellular superoxide dismutase (EC-SOD; SOD3) is a major antioxidant defense in lung and vasculature. A nonsynonomous single nucleotide polymorphism (SNP) in EC-SOD (rs1799895) leads to an arginine to glycine (Arg->Gly) amino acid substitution at position 213 (R213G) in the heparin-binding domain (HBD). In recent human genetic association studies, this SNP attenuates the risk of lung disease, yet paradoxically increases the risk of cardiovascular disease.
Methods and Results
Capitalizing on the complete sequence homology between human and mouse in the HBD, we created an analogous R213G SNP knockin mouse. The R213G SNP did not change enzyme activity, but shifted the distribution of EC-SOD from lung and vascular tissue to extracellular fluid (e.g. bronchoalveolar lavage fluid (BALF) and plasma). This shift reduces susceptibility to lung disease (lipopolysaccharide-induced lung injury) and increases susceptibility to cardiopulmonary disease (chronic hypoxic pulmonary hypertension).
We conclude that EC-SOD provides optimal protection when localized to the compartment subjected to extracellular oxidative stress: thus, the redistribution of EC-SOD from the lung and pulmonary circulation to the extracellular fluids is beneficial in alveolar lung disease but detrimental in pulmonary vascular disease. These findings account for the discrepant risk associated with R213G in humans with lung diseases compared with cardiovascular diseases.
PMCID: PMC4270938  PMID: 25085920
pulmonary hypertension; lung; antioxidant enzymes; cardiovascular disease
2.  The cellular distribution of extracellular superoxide dismutase in macrophages is altered by cellular activation but unaffected by the naturally occurring R213G substitution 
Extracellular superoxide dismutase (EC-SOD) is responsible for the dismutation of the superoxide radical produced in the extracellular space and known to be expressed by inflammatory cells, including macrophages and neutrophils. Here we show that EC-SOD is produced by resting macrophages and associated with the cell surface via the extracellular matrix (ECM)-binding region. Upon cellular activation induced by lipopolysaccharide, EC-SOD is relocated and detected both in the cell culture medium and in lipid raft structures. Although the secreted material presented a significantly reduced ligand-binding capacity, this could not be correlated to proteolytic removal of the ECM-binding region, because the integrity of the material recovered from the medium was comparable to that of the cell surface-associated protein. The naturally occurring R213G amino acid substitution located in the ECM-binding region of EC-SOD is known to affect the binding characteristics of the protein. However, the analysis of macrophages expressing R213G EC-SOD did not present evidence of an altered cellular distribution. Our results suggest that EC-SOD plays a dynamic role in the inflammatory response mounted by activated macrophages.
PMCID: PMC4440334  PMID: 24512907
Antioxidant; Extracellular superoxide dismutase; Inflammation; Macrophage; Free radicals
3.  Co-operation between mast cells and neurons is essential for antigen-mediated bronchoconstriction1 
Mast cells are important sentinels guarding the interface between the environment and the body: a breach in the integrity of this interface can lead to the release of a plethora of mediators which engage the foreign agent, recruit leukocytes, and initiate adaptive physiological changes in the organism. While these capabilities make mast cells critical players in immune defense, it also makes them important contributors to the pathogenesis of diseases such as asthma. Mast cell mediators induce dramatic changes in smooth muscle physiology, and the expression of receptors for these factors by smooth muscle suggests that they act directly to initiate constriction. Contrary to this view, we show here that mast cell-mediated bronchoconstriction is observed only in animals with intact innervation of the lung and that serotonin release alone is required for this action. While ablation of sensory neurons does not limit bronchoconstriction, constriction after antigen challenge is absent in mice in which the cholinergic pathways are compromised. Linking mast cell function to the cholinergic system likely provides an important means of modulating the function of these resident immune cells to physiology of the lung, but may also provide a safeguard against life-threatening anaphylaxis during mast cell degranulation.
PMCID: PMC3901060  PMID: 19494266
Rodent; Mast Cells; Allergy; Lung
4.  Sub-chronic exposure to second hand smoke induces airspace leukocyte infiltration and decreased lung elastance 
Exposure to second hand tobacco smoke is associated with the development and/or exacerbation of several different pulmonary diseases in humans. To better understand the possible effects of second hand smoke exposure in humans, we sub-chronically (4 weeks) exposed mice to a mixture of mainstream and sidestream tobacco smoke at concentrations similar to second hand smoke exposure in humans. The inflammatory response to smoke exposures was assessed at the end of this time by enumeration of pulmonary leukocyte infiltration together with measurements of lung elastance and pathology. This response was measured in both healthy wild type (C57BL/6) mice as well as mouse mutants deficient in the expression of Arhgef1 (Arhgef1−/−) that display constitutive pulmonary inflammation and decreased lung elastance reminiscent of emphysema. The results from this study show that sub-chronic second hand smoke exposure leads to significantly increased numbers of airspace leukocytes in both healthy and mutant animals. While sub-chronic cigarette smoke exposure is not sufficient to induce changes in lung architecture as measured by mean linear intercept, both groups exhibit a significant decrease in lung elastance. Together these data demonstrate that even sub-chronic exposure to second hand smoke is sufficient to induce pulmonary inflammation and decrease lung elastance in both healthy and diseased animals and in the absence of tissue destruction.
PMCID: PMC3429071  PMID: 22934051
second hand smoke; inflammation; lung mechanics
5.  CD38 Plays a Dual Role in Allergen-Induced Airway Hyperresponsiveness 
The multifunctional surface protein CD38 acts as a receptor with ecto-enzymatic activity, hydrolyzing NAD to generate several products known to exhibit Ca2+-mobilizing properties. Although CD38 is a convenient marker of immune cell development, and an indicator of progression for several diseases, it is not restricted to the immune compartment. To determine the potentially multilayered involvement of CD38 in allergen-induced airway inflammation and hyperreactivity, we dissected the potential role of CD38 as a regulator of immunity, but also pulmonary function. CD38-deficient and wild-type (WT) mice were sensitized and airway challenged with ovalbumin, and subsequently analyzed regarding their level of airway hyperresponsiveness (AHR) in response to methacholine. Parameters of lung inflammation were also analyzed. Similar sets of measurements were obtained from reciprocal bone marrow swapping experiments between CD38−/− and WT mice. Mice lacking CD38 exhibit strongly reduced AHR, which is accompanied by a decrease in typical hallmarks of pulmonary inflammation, including eosinophilia and lymphocytic lung infiltrates, as well as Th2-cytokine levels (IL-4, -5, and -13). Antigen-specific immunoglobulin (Ig)E and IgG1 antibody titers are substantially reduced, consistent with CD38 being crucial for mounting a primary humoral systemic immune response. Reconstitution of lethally irradiated, lung-shielded, CD38-deficient mice with WT bone marrow does not restore WT levels of airway hyperreactivity, nor mucus secretion. The opposite experiment, transferring CD38−/− bone marrow into WT mice, also shows reduced AHR levels. These studies demonstrate that CD38 not only acts as a key modulator of the immune response, but also plays an equally important role as an intrinsic pulmonary component.
PMCID: PMC2720120  PMID: 18931329
airway hyperreactivity; pulmonary inflammation; CD38 knockout mouse; bone marrow chimera

Results 1-5 (5)