Todd M. Umstead

Surfactant protein A, an innate immune factor, is expressed in the vaginal mucosa and is present in vaginal lavage fluid

Surfactant protein A (SP‐A), first identified as a component of the lung surfactant system, is now recognized to be an important contributor to host defence mechanisms. SP‐A can facilitate phagocytosis by opsonizing bacteria, fungi and viruses, stimulate the oxidative burst by phagocytes and modulate pro‐inflammatory cytokine production by phagocytic cells. SP‐A can also provide a link between innate and adaptive immune responses by promoting differentiation and chemotaxis of dendritic cells. Because of the obvious relevance of these mechanisms to the host defence and ‘gate keeping’ functions of the lower genital tract, we examined human vaginal mucosa for SP‐A protein and transcripts and analysed vaginal lavage fluid for SP‐A. By immunocytochemistry, SP‐A was identified in two layers of the vaginal epithelium: the deep intermediate layer (the site of newly differentiated epithelial cells); and the superficial layer (comprising dead epithelial cells), where SP‐A is probably extracellular and associated with a glycocalyx. Transcripts of SP‐A were identified by Northern blot analysis in RNA isolated from vaginal wall and shown, by sequencing of reverse transcription–polymerase chain reaction products, to be derived from each of the two closely related SP‐A genes, SP‐A1 and SP‐A2. SP‐A was identified in vaginal lavage fluid by two‐dimensional gel electrophoresis, and confirmed by mass spectrometry. This study provides evidence, for the first time, that SP‐A is produced in a squamous epithelium, namely the vaginal mucosa, and has a localization that would allow it to contribute to both the innate and adaptive immune response. The findings support the hypothesis that in the vagina, as in lung, SP‐A is an essential component of the host‐defence system. A corollary hypothesis is that qualitative and quantitative alterations of normal SP‐A may play a role in the pathogenesis of lower genital tract inflammatory conditions.

Surfactant protein A2 (SP-A2) variants expressed in CHO cells stimulate phagocytosis of Pseudomonas aeruginosa more than do SP-A1 variants

ABSTRACT Surfactant protein A (SP-A) enhances phagocytosis of Pseudomonas aeruginosa. Two functional genes, SP-A1 and SP-A2, encode human SP-A. As we showed before, baculovirus-mediated insect cell-expressed SP-A2 enhances the association of P. aeruginosa with rat alveolar macrophages (rAMs) more than does SP-A1. However, true phagocytosis (internalization) was not shown, and insect cell derived proteins lack or are defective in certain mammalian posttranslational modifications that may be important for SP-A1 and SP-A2 activity and specificity. Here we used SP-A1 (6A2, 6A4) and SP-A2 (1A0, 1A1) allele variants expressed by CHO (Chinese hamster ovary) mammalian cells to study their effect on association and/or internalization of P. aeruginosa by rAMs and/or human AMs (hAMs) and to study if phagocytosis can be modulated differentially and/or more effectively by CHO cell-expressed SP-A variants than by insect-cell expressed SP-A variants. For cell association and internalization assessments, light microscopy and fluorescence-activated cell sorter analyses were used, respectively. We found the following for the first time. (i) SP-A2 variants enhanced phagocytosis (cell association and/or internalization) of P. aeruginosa more than SP-A1 variants did, and the cell association correlated with internalization. (ii) Differences in the activities of SP-A variants were observed in the following order: 1A1>1A0>6A2>6A4. (iii) rAMs, although more active than hAMs, are an appropriate model, as SP-A2 variants exhibited activity higher than that seen for SP-A1 variants with either rAMs or hAMs. (iv) CHO cell-expressed SP-A was considerably more active than insect cell-expressed variants. We conclude that SP-A2 variants stimulate phagocytosis of P. aeruginosa more effectively than SP-A1 variants and that posttranslational modifications positively influence the phagocytic activity of SP-A.

Proteomic profiling of human plasma by iTRAQ reveals down-regulation of ITI-HC3 and VDBP by cigarette smoking.

Biomarkers in noninvasive fluids indicative of cigarette smokes effects are urgently needed. In this pilot study, we utilized the proteomic approach, isobaric Tags for Relative and Absolute Quantitation (iTRAQ), to identify differentially expressed plasma proteins in healthy cigarette smokers compared to healthy nonsmokers; select proteins were further confirmed by immunoblot analysis. Significant, differentially expressed proteins identified in the plasma separated subjects based on their condition as smokers or nonsmokers. Several of the proteins identified in this study are associated with immunity and inflammatory responses and have been shown to be associated with tobacco-related diseases, including chronic obstructive pulmonary disease (COPD) and lung cancer. Proteins up-regulated in smokers included complement component 8 polypeptide chains α, β, and γ, and mannose-binding protein C, and proteins down-regulated included inter-α-trypsin inhibitor heavy chain H3 (ITI-HC3) and vitamin D-binding protein (VDBP). In addition, gelsolin and vitronectin, known tissue leakage proteins, were up- and down-regulated, respectively. Our results demonstrate for the first time that chronic cigarette smoking can influence the expression profile of the human plasma proteome. Proteins identified in this pilot study may serve as candidate biomarkers of diseases resulting from exposure to cigarette smoke in future molecular epidemiological studies.

Sex differences in the impact of ozone on survival and alveolar macrophage function of mice after Klebsiella pneumoniae infection

BackgroundSex differences have been described in a number of pulmonary diseases. However, the impact of ozone exposure followed by pneumonia infection on sex-related survival and macrophage function have not been reported. The purpose of this study was to determine whether ozone exposure differentially affects: 1) survival of male and female mice infected with Klebsiella pneumoniae, and 2) the phagocytic ability of macrophages from these mice.MethodsMale and female C57BL/6 mice were exposed to O3 or to filtered air (FA) (control) and then infected intratracheally with K. pneumoniae bacteria. Survival was monitored over a 14-day period, and the ability of alveolar macrophages to phagocytize the pathogen in vivo was investigated after 1 h.Results1) Both male and female mice exposed to O3 are significantly more susceptible to K. pneumoniae infection than mice treated with FA; 2) although females appeared to be more resistant to K. pneumoniae than males, O3 exposure significantly increased the susceptibility of females to K. pneumoniae infection to a greater degree than males; 3) alveolar macrophages from O3-exposed male and female mice have impaired phagocytic ability compared to macrophages from FA-exposed mice; and 4) the O3-dependent reduction in phagocytic ability is greater in female mice.ConclusionO3 exposure reduces the ability of mice to survive K. pneumoniae infection and the reduced phagocytic ability of alveolar macrophages may be one of the contributing factors. Both events are significantly more pronounced in female mice following exposure to the environmental pollutant, ozone.

Age-related changes in the expression and oxidation of bronchoalveolar lavage proteins in the rat

The incidence and severity of many lung diseases change with age. Some diseases, such as pneumonia, occur with increased frequency in children and the elderly. Proteins obtained by bronchoalveolar lavage (BAL) serve as the first line of defense against inhaled toxins and pathogens. Age-related changes in BAL protein expression and oxidative modification were examined in juvenile (1 mo), young adult (2 mo), and aged (18 mo) F344 rats using two-dimensional difference gel electrophoresis (2D-DIGE), matrix-assisted laser desorption ionization-time of flight/time of flight (MALDI-ToF/ToF) tandem mass spectrometry, and carbonyl immunoblotting. Using 2D-DIGE, we detected 563 protein spots, and MALDI-ToF/ToF identified 204 spots comprising 31 proteins; 21 changed significantly (17 increases) between juvenile and young adult or aged rats, but for 12 of these proteins, levels had a biphasic pattern, and levels in aged rats were less than in young adults. Relative carbonylation was determined by comparison of immunostaining with total protein staining on each oxidized protein blot. We found that aged rats had significantly increased oxidation in 13 proteins compared with juvenile rats. Many of the proteins altered in expression or oxidation level had functions in host defense, redox regulation, and protein metabolism. We speculate that low levels of expression of host defense proteins in juvenile rats and decreases in levels of these proteins between young adult and aged rats may predispose these groups to pneumonia. In addition, we have shown age-related increases in protein oxidation that may compromise host defense function in aged rats.

Down-regulation of 14-3-3 isoforms and annexin A5 proteins in lung adenocarcinoma induced by the tobacco-specific nitrosamine NNK in the A/J mouse revealed by proteomic analysis.

The tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a potent lung carcinogen in the A/J mouse model. Here we identified and validated, using two-dimensional difference gel electrophoresis (2D-DIGE) coupled with mass spectrometry and immunoblotting, proteins that are differentially expressed in the lungs of mice treated with NNK versus vehicle control treatment. We also determined whether protein levels in the lungs of NNK-treated mice could be further modulated by the chemopreventive agent 1,4-phenylenebis(methylene)selenocyanate (p-XSC). The proteins identified in this study are SEC14-like 3, dihydropyrimidinase-like 2, proteasome subunit alpha type 5, annexin A5, 14-3-3 protein isoforms (theta, epsilon, sigma, and zeta), Rho GDP dissociation inhibitor alpha, myosin light polypeptide 6, tubulin-alpha-1, vimentin, Atp5b protein, alpha-1-antitrypsin, and Clara cell 10 kDa protein (CC10). Among those proteins, we demonstrated for the first time that 14-3-3 isoforms (theta, epsilon, and sigma) and annexin A5 were significantly down-regulated in mouse lung adenocarcinoma induced by NNK and were recovered by p-XSC. These proteins are involved in a variety of biological functions that are critical in lung carcinogenesis. Identification of these proteins in surrogate tissue in future studies would be highly useful in early detection of lung adenocarcinoma and clinical chemoprevention trials.

In Vitro Exposure of Proteins to Ozone

An in vitro system has been developed to expose proteins to ozone. The system is designed to deliver consistent and accurate levels of ozone over a range of concentrations (between 0.1 and ≥10 ppm) with extended exposure times (24 h or longer) in a humidified environment (100%). In the experiment presented in this article, ozone concentrations between 0.1 and 2.0 ppm were used. Ozone was generated by an electrical discharge ozonizer to ensure stability; it was continually monitored by an ultraviolet ozone analyzer and was precisely controlled by mass flow controllers, which gave reproducible results between runs. Humidity was closely regulated in the system to allow small amounts of protein solutions (50 μL or less) to be exposed without significant changes (<0.2%) in sample volume. The degree of surfactant protein-A (SP-A) oxidation by ozone was measured between runs to demonstrate the reproducibility of the system. A detailed description of the system is given, and protein oxidation detection methods and their limitations are discussed. Using these methods, we were able to assess oxidation of SP-A that apparently occurred prior to its isolation from the lung by bronchoalveolar lavage. This in vitro system allowed us to expose small amounts of protein to ozone in a simple, highly controlled, and reproducible manner.

An in vitro cell model system for the study of the effects of ozone and other gaseous agents on phagocytic cells.

Ozone (O(3)), a major component of air pollution and a very strong oxidizing agent, can lead to lung injury associated with edema, inflammation, and epithelial cell damage. The ozone effects on pulmonary immune cells have been studied by various in vivo and in vitro systems. In this report, we characterized a model system of cells of monocyte/macrophage lineage (THP-1 cells) exposed to ozone in vitro by studying cell viability and cell surface marker expression. THP-1 cells exposed to ozone in concentrations ranging from 0.1 to 0.5 ppm for 1 h were analyzed for cell viability and apoptosis (Annexin V/7-Amino-actinomycin D (7-AAD) flow cytometric assay) either immediately after ozone exposure or at later time points. This analysis showed absence of apoptosis and a small decrease in cell viability (5-17%) in ozone-exposed THP-1 cells. Cell surface protein expression (CD14 and CD11b) did not change following ozone exposure, but the effect of lipopolysaccharride (LPS) on TNF-alpha production following ozone exposure changed compared to filtered air/LPS-exposed cells. These findings indicate that this in vitro ozone cell-exposure system may be used in studies where the effects of various agents (physiological and non-physiological) on phagocytic cells can be analyzed. This model system offers conditions where the experimental results are not due to cell death, but rather due to the effects of ozone and/or agents under investigation.

Cyclic Changes in the Level of the Innate Immune Molecule, Surfactant Protein-A, and Cytokines in Vaginal Fluid

Our knowledge of the innate host defenses in the vagina, a site where these defenses are essential to protecting the host upper reproductive tract from invasion by pathogens, is as yet rudimentary. Specifically, little is known about the pattern‐recognition component of vaginal innate immunity, the relationship of pattern‐recognition molecules to known cytokine levels, and the role of gonadal hormones in their regulation.

Dual-Platform Proteomics Study of Plasma Biomarkers in Pediatric Patients Undergoing Cardiopulmonary Bypass

Plasma samples from pediatric cardiac patients undergoing cardiopulmonary bypass (CPB) procedures were used to identify and characterize patterns of changes in potential biomarkers related to tissue damage and inflammation. These included proteins associated with systemic inflammatory response syndrome. Potential biomarkers were identified using a dual-platform proteomics approach requiring ∼150 μL of plasma, which included two-dimensional difference gel electrophoresis (2D-DIGE) and a multiplexed immunoassay. Methods used in the dual approach measured levels of 129 proteins in plasma from pediatric CPB patients. Of these, 70 proteins changed significantly (p < 0.05) between time points, and 36 of these retained significance after the highly stringent Bonferroni correction [p < 0.001 for 2D-DIGE and p < 0.00056 for multianalyte profile (MAP) assays]. Many of the changing proteins were associated with tissue damage, inflammation, and oxidative stress. This study uses a novel approach that combines two discovery proteomics techniques to identify a pattern of potential biomarkers changing after CPB. This approach required only 150 μL of plasma per time point and provided quantitative information on 129 proteins. The changes in levels of expression of these proteins may provide insight into the understanding, treatment, and prevention of systemic inflammation, thereby helping to improve the outcomes of pediatric CPB patients.