The effect of lipoprotein-associated phospholipase A2 deficiency on pulmonary allergic responses in Aspergillus fumigatus sensitized mice.

BackgroundLipoprotein-associated phospholipase A2 (Lp-PLA2)/platelet-activating factor acetylhydrolase (PAF-AH) has been implicated in the pathogenesis of cardiovascular disease. A therapeutic targeting of this enzyme was challenged by the concern that increased circulating platelet activating factor (PAF) may predispose to or increase the severity of the allergic airway response. The aim of this study was to investigate whether Lp-PLA2 gene deficiency increases the risk of PAF and IgE-mediated inflammatory responses in vitro and in vivo using mouse models.MethodsLp-PLA2-/- mice were generated and back crossed to the C57BL/6 background. PAF-AH activity was measured using a hydrolysis assay in serum and bronchoalveolar lavage (BAL) samples obtained from mice. Aspergillus fumigatus (Af)-specific serum was prepared for passive allergic sensitization of mice in vivo and mast cells in vitro. β- hexosaminidase release was studied in bone marrow derived mast cells sensitized with Af-specific serum or DNP-IgE and challenged with Af or DNP, respectively. Mice were treated with lipopolysaccharide (LPS) and PAF intratracheally and studied 24 hours later. Mice were sensitized either passively or actively against Af and were studied 48 hours after a single intranasal Af challenge. Airway responsiveness to methacholine, inflammatory cell influx in the lung tissue and BAL, immunoglobulin (ELISA) and cytokine (Luminex) profiles were compared between the wild type (WT) and Lp-PLA2-/- mice.ResultsPAF-AH activity was reduced but not completely abolished in Lp-PLA2-/- serum or by in vitro treatment of serum samples with a high saturating concentration of the selective Lp-PLA2 inhibitor, SB-435495. PAF inhalation significantly enhanced airway inflammation of LPS treated WT and Lp-PLA2-/- mice to a similar extent. Sensitized WT and Lp-PLA2-/- bone-marrow derived mast cells released β-hexosaminidase following stimulation by allergen or IgE crosslinking to equivalent levels. Wild type and Lp-PLA2-/- mice responded to passive or active allergic sensitization by significant IgE production, airway inflammation and hyperresponsiveness after Af challenge. BAL cell influx was not different between these strains while IL-4, IL-5, IL-6 and eotaxin release was attenuated in Lp-PLA2-/- mice. There were no differences in the amount of total IgE levels in the Af sensitized WT and Lp-PLA2-/- mice.ConclusionsWe conclude that Lp-PLA2 deficiency in C57BL/6 mice did not result in a heightened airway inflammation or hyperresponsiveness after PAF/LPS treatment or passive or active allergic sensitization and challenge

Role of pathogenic oral flora in postoperative pneumonia following brain surgery

Background: Post-operative pulmonary infection often appears to result from aspiration of pathogens colonizing the oral cavity. It was hypothesized that impaired periodontal status and pathogenic oral bacteria significantly contribute to development of aspiration pneumonia following neurosurgical operations. Further, the prophylactic effects of a single dose preoperative cefazolin on the oral bacteria were investigated. Methods: A matched cohort of 18 patients without postoperative lung complications was compared to 5 patients who developed pneumonia within 48 hours after brain surgery. Patients waiting for elective operation of a single brain tumor underwent dental examination and saliva collection before surgery. Bacteria from saliva cultures were isolated and periodontal disease was scored according to type and severity. Patients received 15 mg/kg cefazolin intravenously at the beginning of surgery. Serum, saliva and bronchial secretion were collected promptly after the operation. The minimal inhibitory concentrations of cefazolin regarding the isolated bacteria were determined. The actual antibiotic concentrations in serum, saliva and bronchial secretion were measured by capillary electrophoresis upon completion of surgery. Bacteria were isolated again from the sputum of postoperative pneumonia patients. Results: The number and severity of coexisting periodontal diseases were significantly greater in patients with postoperative pneumonia in comparison to the control group (p = 0.031 and p = 0.002, respectively). The relative risk of developing postoperative pneumonia in high periodontal score patients was 3.5 greater than in patients who had low periodontal score (p < 0.0001). Cefazolin concentration in saliva and bronchial secretion remained below detectable levels in every patient. Conclusion: Presence of multiple periodontal diseases and pathogenic bacteria in the saliva are important predisposing factors of postoperative aspiration pneumonia in patients after brain surgery. The low penetration rate of cefazolin into the saliva indicates that its prophylactic administration may not be sufficient to prevent postoperative aspiration pneumonia. Our study suggests that dental examination may be warranted in order to identify patients at high risk of developing postoperative respiratory infections

Attenuated allergic airway hyperresponsiveness in C57BL/6 mice is associated with enhanced surfactant protein (SP)-D production following allergic sensitization

BACKGROUND: C57BL/6 mice have attenuated allergic airway hyperresponsiveness (AHR) when compared with Balb/c mice but the underlying mechanisms remain unclear. SP-D, an innate immune molecule with potent immunosuppressive activities may have an important modulatory role in the allergic airway response and the consequent physiological changes. We hypothesized that an elevated SP-D production is associated with the impaired ability of C57BL/6 mice to develop allergic AHR. METHODS: SP-D mRNA and protein expression was investigated during development of allergic airway changes in a model of Aspergillus fumigatus (Af)-induced allergic inflammation. To study whether strain dependency of allergic AHR is associated with different levels of SP-D in the lung, Balb/c and C57BL/6 mice were compared. RESULTS: Sensitization and exposure to Af induced significant airway inflammation in both mouse strains in comparison with naïve controls. AHR to acetylcholine however was significantly attenuated in C57BL/6 mice in spite of increased eosinophilia and serum IgE when compared with Balb/c mice (p < 0.05). Af challenge of sensitized C57BL/6 mice induced a markedly increased SP-D protein expression in the SA surfactant fraction (1,894 ± 170% of naïve controls) that was 1.5 fold greater than the increase in Balb/c mice (1,234 ± 121% p < 0.01). These changes were selective since levels of the hydrophobic SP-B and SP-C and the hydrophilic SP-A were significantly decreased following sensitization and challenge with Af in both strains. Further, sensitized and exposed C57BL/6 mice had significantly lower IL-4 and IL-5 in the BAL fluid than that of Balb/c mice (p < 0.05). CONCLUSIONS: These results suggest that enhanced SP-D production in the lung of C57BL/6 mice may contribute to an attenuated AHR in response to allergic airway sensitization. SP-D may act by inhibiting synthesis of Th2 cytokines

Chronic obstructive pulmonary disease and inhaled steroids alter surfactant protein D (SP-D) levels: a cross-sectional study

<p>Abstract</p> <p>Background</p> <p>Surfactant protein D (SP-D), an innate immune molecule, plays an important protective role during airway inflammation. Deficiency of this molecule induces emphysematous changes in murine lungs, but its significance in human COPD remains unclear.</p> <p>Methods</p> <p>We collected bronchoalveolar lavage fluid from 20 subjects with varying degrees of COPD (8 former smokers and 12 current smokers) and 15 asymptomatic healthy control subjects (5 never smokers, 3 remote former smokers, and 7 current smokers). All subjects underwent a complete medical history and pulmonary function testing. SP-D was measured by Enzyme-Linked ImmunoSorbent Assay. Statistical analysis was performed using nonparametric methods and multivariable linear regression for control of confounding. The effect of corticosteroid treatment on SP-D synthesis was studied <it>in vitro </it>using an established model of isolated type II alveolar epithelial cell culture.</p> <p>Results</p> <p>Among former smokers, those with COPD had significantly lower SP-D levels than healthy subjects (median 502 and 1067 ng/mL, respectively, p = 0.01). In a multivariable linear regression model controlling for age, sex, race, and pack-years of tobacco, COPD was independently associated with lower SP-D levels (model coefficient -539, p = 0.04) and inhaled corticosteroid use was independently associated with higher SP-D levels (398, p = 0.046). To support the hypothesis that corticosteroids increase SP-D production we used type II alveolar epithelial cells isolated from adult rat lungs. These cells responded to dexamethasone treatment by a significant increase of SP-D mRNA (p = 0.041) and protein (p = 0.037) production after 4 days of culture.</p> <p>Conclusion</p> <p>Among former smokers, COPD is associated with lower levels of SP-D and inhaled corticosteroid use is associated with higher levels of SP-D in the lung. Dexamethasone induced SP-D mRNA and protein expression in isolated epithelial cells <it>in vitro</it>. Given the importance of this molecule as a modulator of innate immunity and inflammation in the lung, low levels may play a role in the pathogenesis and/or progression of COPD. Further, we speculate that inhaled steroids may induce SP-D expression and that this mechanism may contribute to their beneficial effects in COPD. Larger, prospective studies are warranted to further elucidate the role of surfactant protein D in modulating pulmonary inflammation and COPD pathogenesis.</p

Dissociation by steroids of eosinophilic inflammation from airway hyperresponsiveness in murine airways

BACKGROUND: The link between eosinophils and the development of airway hyperresponsiveness (AHR) in asthma is still controversial. This question was assessed in a murine model of asthma in which we performed a dose ranging study to establish whether the dose of steroid needed to inhibit the eosinophil infiltration correlated with that needed to block AHR. METHODS: The sensitised BALB/c mice were dosed with vehicle or dexamethasone (0.01–3 mg/kg) 2 hours before and 6 hours after each challenge (once daily for 6 days) and 2 hours before AHR determination by whole-body plethysmography. At 30 minutes after the AHR to aerosolised methacholine the mice were lavaged and differential white cell counts were determined. Challenging with antigen caused a significant increase in eosinophils in the bronchoalveolar lavage (BAL) fluid and lung tissue, and increased AHR. RESULTS: Dexamethasone reduced BAL and lung tissue eosinophilia (ED(50 )values of 0.06 and 0.08 mg/kg, respectively), whereas a higher dose was needed to block AHR (ED(50 )of 0.32 mg/kg at 3 mg/ml methacholine. Dissociation was observed between the dose of steroid needed to affect AHR in comparison with eosinophilia and suggests that AHR is not a direct consequence of eosinophilia. CONCLUSION: This novel pharmacological approach has revealed a clear dissociation between eosinophilia and AHR by using steroids that are the mainstay of asthma therapy. These data suggest that eosinophilia is not associated with AHR and questions the rationale that many pharmaceutical companies are adopting in developing low-molecular-mass compounds that target eosinophil activation/recruitment for the treatment of asthma

Downregulation of Integrin β4 Decreases the Ability of Airway Epithelial Cells to Present Antigens

Airway epithelial cells have been demonstrated to be accessory antigen presentation cells (APC) capable of activating T cells and may play an important role in the development of allergic airway inflammation of asthma. In asthmatic airways, loss of expression of the adhesion molecule integrin β4 (ITGB4) and an increase in Th2 inflammation bias has been observed in our previous study. Given that ITGB4 is engaged in multiple signaling pathways, we studied whether disruption of ITGB4-mediated cell adhesion may contribute to the adaptive immune response of epithelial cells, including their ability to present antigens, induce the activate and differentiate of T cells. We silenced ITGB4 expression in bronchial epithelial cells with an effective siRNA vector and studied the effects of ITGB4 silencing on the antigen presentation ability of airway epithelial cells. T cell proliferation and cytokine production was investigated after co-culturing with ITGB4-silenced epithelial cells. Surface expression of B7 homologs and the major histocompatibility complex (MHC) class II was also detected after ITGB4 was silenced. Our results demonstrated that silencing of ITGB4 resulted in impaired antigen presentation processes and suppressed T cell proliferation. Meanwhile, decrease in Th1 cytokine production and increase in Th17 cytokine production was induced after co-culturing with ITGB4-silenced epithelial cells. Moreover, HLA-DR was decreased and the B7 homologs expression was different after ITGB4 silencing. Overall, this study suggested that downregulation of ITGB4 expression in airway epithelial cells could impair the antigen presentation ability of these cells, which further regulate airway inflammation reaction in allergic asthma

Susceptibility to ozone-induced airway inflammation is associated with decreased levels of surfactant protein D

BACKGROUND: Ozone (O(3)), a common air pollutant, induces exacerbation of asthma and chronic obstructive pulmonary disease. Pulmonary surfactant protein (SP)-D modulates immune and inflammatory responses in the lung. We have shown previously that SP-D plays a protective role in a mouse model of allergic airway inflammation. Here we studied the role and regulation of SP-D in O(3)-induced inflammatory changes in the lung. METHODS: To evaluate the effects of O(3 )exposure in mouse strains with genetically different expression levels of SP-D we exposed Balb/c, C57BL/6 and SP-D knockout mice to O(3 )or air. BAL cellular and cytokine content and SP-D levels were evaluated and compared between the different strains. The kinetics of SP-D production and inflammatory parameters were studied at 0, 2, 6, 12, 24, 48, and 72 hrs after O(3 )exposure. The effect of IL-6, an O(3)-inducible cytokine, on the expression of SP-D was investigated in vitro using a primary alveolar type II cell culture. RESULTS: Ozone-exposed Balb/c mice demonstrated significantly enhanced acute inflammatory changes including recruitment of inflammatory cells and release of KC and IL-12p70 when compared with age- and sex-matched C57BL/6 mice. On the other hand, C57BL/6 mice had significantly higher levels of SP-D and released more IL-10 and IL-6. Increase in SP-D production coincided with the resolution of inflammatory changes. Mice deficient in SP-D had significantly higher numbers of inflammatory cells when compared to controls supporting the notion that SP-D has an anti-inflammatory function in our model of O(3 )exposure. IL-6, which was highly up-regulated in O(3 )exposed mice, was capable of inducing the expression of SP-D in vitro in a dose dependent manner. CONCLUSION: Our data suggest that IL-6 contributes to the up-regulation of SP-D after acute O(3 )exposure and elevation of SP-D in the lung is associated with the resolution of inflammation. Absence or low levels of SP-D predispose to enhanced inflammatory changes following acute oxidative stress

Glucocorticoid receptor gene polymorphisms associated with progression of lung disease in young patients with cystic fibrosis

<p>Abstract</p> <p>Background</p> <p>The variability in the inflammatory burden of the lung in cystic fibrosis (CF) patients together with the variable effect of glucocorticoid treatment led us to hypothesize that <it>glucocorticoid receptor </it>(<it>GR</it>) gene polymorphisms may affect glucocorticoid sensitivity in CF and, consequently, may contribute to variations in the inflammatory response.</p> <p>Methods</p> <p>We evaluated the association between four <it>GR </it>gene polymorphisms, <it>TthIII</it>, <it>ER22/23EK</it>, <it>N363S </it>and <it>BclI</it>, and disease progression in a cohort of 255 young patients with CF. Genotypes were tested for association with changes in lung function tests, infection with <it>Pseudomonas aeruginosa </it>and nutritional status by multivariable analysis.</p> <p>Results</p> <p>A significant non-corrected for multiple tests association was found between <it>BclI </it>genotypes and decline in lung function measured as the forced expiratory volume in one second (FEV₁) and the forced vital capacity (FVC). Deterioration in FEV₁and FVC was more pronounced in patients with the <it>BclI </it>GG genotype compared to the group of patients with <it>BclI </it>CG and CC genotypes (p = 0.02 and p = 0.04 respectively for the entire cohort and p = 0.01 and p = 0.02 respectively for F508del homozygous patients).</p> <p>Conclusion</p> <p>The <it>BclI </it>polymorphism may modulate the inflammatory burden in the CF lung and in this way influence progression of lung function.</p

B Cell Antigen Presentation Promotes Th2 Responses and Immunopathology during Chronic Allergic Lung Disease

Background: The role of B cells in allergic asthma remains undefined. One mechanism by which B cells clearly contribute to allergic disease is via the production of specific immunoglobulin, and especially IgE. Cognate interactions with specific T cells result in T cell help for B cells, resulting in differentiation and immunoglobulin secretion. Proximal to (and required for) T cell-dependent immunoglobulin production, however, is antigen presentation by B cells. While interaction with T cells clearly has implications for B cell function and differentiation, this study investigated the role that B cells have in shaping the T cell response during chronic allergic lung disease. Methodology/Principal Findings: In these studies, we used a clinically relevant mouse model of chronic allergic lung disease to study the role of B cells and B cell antigen presentation in this disease. In these studies we present several novel findings: 1) Lung B cells from chronically allergen challenged mice up-regulated MHC II and costimulatory molecules CD40, CD80 and CD86. 2) Using in vitro studies, B cells from the lungs of allergen challenged mice could present antigen to T cells, as assessed by T cell proliferation and the preferential production of Th2 cytokines. 3) Following chronic allergen challenge, the levels of Th2 cytokines IL-4 and IL-5 in the lungs and airways were significantly attenuated in B cell 2/2 mice, relative to controls. 4) B cell driven Th2 responses and mucus hyper secretion in the lungs were dependent upon MHC II expression by B cells. Conclusions/Significance: Collectively, these results provide evidence for antigen presentation as a novel mechanism b