James M. Stark

Surfactant protein-A enhances respiratory syncytial virus clearance in vivo

To determine the role of surfactant protein-A(SP-A) in antiviral host defense, mice lacking SP-A (SP-A-/-) were produced by targeted gene inactivation. SP-A-/- and control mice (SP-A+/+) were infected with respiratory syncytial virus (RSV) by intratracheal instillation. Pulmonary infiltration after infection was more severe in SP-A-/- than in SP-A+/+ mice and was associated with increased RSV plaque-forming units in lung homogenates. Pulmonary infiltration with polymorphonuclear leukocytes was greater in the SP-A-/- mice. Levels of proinflammatory cytokines tumor necrosis factor-alpha and interleukin-6 were enhanced in lungs of SP-A-/- mice. After RSV infection, superoxide and hydrogen peroxide generation was deficient in macrophages from SP-A-/- mice, demonstrating a critical role of SP-A in oxidant production associated with RSV infection. Coadministration of RSV with exogenous SP-A reduced viral titers and inflammatory cells in the lung of SP-A-/- mice. These findings demonstrate that SP-A plays an important host defense role against RSV in vivo.

Clara Cell Secretory Protein Modulates Lung Inflammatory and Immune Responses to Respiratory Syncytial Virus Infection

Clara cell secretory protein (CCSP) has been shown to have anti-inflammatory and immunomodulatory functions in the lung. Respiratory syncytial virus (RSV) is the most common cause of respiratory infection in infants and young children. RSV usually infects small airways and likely interacts with the Clara cells of bronchioles. To determine a possible role for CCSP during acute RSV infection, CCSP-deficient (CCSP−/−) and wild-type (WT) mice were intratracheally infected with RSV and the lung inflammatory and immune responses to RSV infection were assessed. RSV-F gene expression was increased in the lungs of CCSP−/− mice as compared with WT mice following RSV infection, consistent with increased viral persistence. Lung inflammation was significantly increased in CCSP−/− mice as compared with WT mice after infection. Moreover, although the levels of Th1 cytokines were similar, the levels of Th2 cytokines and neutrophil chemokines were increased in the lungs of CCSP−/− mice following infection. Physiologic endpoints of exacerbated lung disease, specifically airway reactivity and mucus production, were increased in CCSP−/− mice after RSV infection. Importantly, restoration of CCSP in the airways of CCSP−/− mice abrogated the increased viral persistence, lung inflammation, and airway reactivity. These findings suggest a role for CCSP and Clara cells in regulating lung inflammatory and immune responses to RSV infection.

Use of a remifentanil–propofol mixture for pediatric flexible fiberoptic bronchoscopy sedation

Background : Flexible fiberoptic bronchoscopy is an important diagnostic tool for pediatric pulmonologists. Because of its favorable respiratory profile, ketamine has become a popular sedative for this procedure, but may be associated with unpleasant emergence reactions in the older child. Remifentanil is a newer, ultra‐short acting opioid that has been shown to provide effective sedation and cough suppression for fiberoptic bronchoscopy when combined with intermittent propofol boluses. However, delivery of these agents as a combined, single infusion has not been described.

Heat shock inhibits TNF-induced ICAM-1 expression in human endothelial cells via I kappa kinase inhibition

The pulmonary vascular endothelium plays a critical role in lung inflammation. As a result of proinflammatory cytokine expression, adhesion molecules are upregulated on the surface of the endothelial cells. Adhesion molecules facilitate recruitment of leukocytes and thus, have been targeted for potential anti-inflammatory strategies. Prior induction of the stress response through thermal stimulation, or heat shock, alters proinflammatory gene expression by attenuating NF-&kgr;B signaling. As intercellular adhesion molecule-(ICAM) 1 expression is, in part, NF-&kgr;B-dependent, we hypothesized that heat shock would inhibit ICAM-1 expression. Heat shocking endothelial cells resulted in heat shock protein (HSP) expression as measured by HSP-70 induction, and decreased TNF-&agr;-induced ICAM-1 expression in a manner that appeared to be transcriptionally mediated. Following heat shock, decreased TNF-&agr;-induced NF-&kgr;B activation was observed and was associated with preservation of I&kgr;B-&agr; and a decrease in phosphorylated I&kgr;B-&agr; that correlated to inhibition of I kappa kinase (IKK) activity. Interestingly, exposing respiratory epithelial cells to heat shock, which results in NF-&kgr;B inhibition, did not affect TNF-induced ICAM-1 expression. We conclude that heat shock decreases endothelial cell ICAM-1 expression via inhibition of IKK activity.

The impact of respiratory infections on asthma.

It is apparent that the effects of viral respiratory infections on the development of airway hyperresponsiveness are multiple and interrelated and involved the production of viral specific IgE, upregulation of leukocyte inflammatory activity, enhancement of the factors involved in the generation of late phase allergic responses, altered beta-adrenergic and cholinergic nervous system activity, and damage to the airway epithelium. The summation of these effects is the development of airway inflammation rather than a direct effect on bronchial smooth muscle, per se. An understanding of this pathogenesis underscores the relative importance of anti-inflammatory rather than antimicrobial therapy in viral-induced exacerbations in asthma symptoms.

Immune and Functional Role of Nitric Oxide in a Mouse Model of Respiratory Syncytial Virus Infection

BACKGROUND Respiratory syncytial virus (RSV) infection of respiratory epithelial cell cultures increases expression of inducible nitric oxide synthase (iNOS). The present study was designed to evaluate both the effect of RSV infection on expression of iNOS and the role of NO in the host responses to RSV infection in vivo. METHODS RSV infection was performed by nasal inoculation of BALB/c mice (6-8 weeks old). Total cell and differential counts were measured in bronchoalveolar lavage (BAL) fluid. Lung nitrates were measured in BAL fluid by use of the Greiss reaction, and cytokines were measured by enzyme-linked immunosorbent assay. Lung hyperresponsiveness to methacholine was measured by use of a Buxco unrestrained whole-body plethysmograph. RESULTS RSV infection increased levels of lung nitrites, levels of iNOS protein and activity, and levels of iNOS mRNA. RSV infection resulted in recruitment of neutrophils and lymphocytes into the lungs, enhanced levels of interferon (IFN)-gamma, and increased airway hyperresponsiveness (AHR). Treatment with iNOS inhibitors (2-amino-5,6-dihydro-6-methyl-4H-1,3-thiazine and N-nitro-L-arginine methyl ester) increased RSV titers in the lungs yet reduced lung inflammation and RSV-induced AHR. Inhibition of iNOS activity with either agent did not significantly alter levels of IFN-gamma or interleukin-4 in the lungs. CONCLUSIONS The results of the present study suggest that RSV-induced production of NO participates in complex host responses and may mediate important aspects of the clinical disease.

Molecular Analysis of CCR-3 Events in Eosinophilic Cells

CCR-3 is a major receptor involved in regulating eosinophil trafficking. Initial analysis of chemokine receptors has demonstrated unique receptor events in different cell types, indicating the importance of investigating CCR-3 events in eosinophilic cell lines. We now report that the eosinophilic cell line, acute myelogenous leukemia (AML) 14.3D10, expresses eosinophil granule proteins and eotaxin, but has no detectable expression of eosinophil chemokine receptors. Treatment of the cell line with butyric acid and IL-5 results in a dose-dependent synergistic induction of CCR-3 and, to a lesser extent, CCR-1 and CCR-5. Interestingly, using a luciferase reporter construct under the control of the hCCR-3 promoter, the uninduced and induced cells display high, but comparable, levels of promoter activity. Differentiated AML cells developed enhanced functional activation, as indicated by adhesion to respiratory epithelial cells and chemokine-induced transepithelial migration. Chemokine signaling did not inhibit adenylate cyclase activity even though calcium transients were blocked by pertussis toxin. Additionally, chemokine-induced calcium transients were inhibited by pretreatment with PMA, but not forskolin. Eotaxin treatment of differentiated AML cells resulted in marked down-modulation of CCR-3 expression for at least 18 h. Receptor internalization was not dependent upon chronic ligand exposure and was not accompanied by receptor degradation. Thus, CCR-3 is a late differentiation marker on AML cells and uses a signal transduction pathway involving rapid and prolonged receptor internalization, calcium transients inhibitable by protein kinase C but not protein kinase A, and the paradoxical lack of inhibition of adenylate cyclase activity.

Detection of enhanced neutrophil adhesion to parainfluenza-infected airway epithelial cells using a modified myeloperoxidase assay in a microtiter format

Despite growing evidence that respiratory virus infections precipitate episodes of airway obstruction and airway hyper-responsiveness in young children and in asthma, little information is available on the mechanisms by which virus infections alter the airway physiology. Airway inflammatory changes (including influx of inflammatory cells such as neutrophils) have been described during episodes of airway hyper-responsiveness in both animal models and human subjects. Neutrophil damage to several cell types has been shown to require adhesion as a primary step. In order to examine the potential interactions between virus-infected airway epithelial cells and neutrophils, we have studied the ability of neutrophils to adhere to virus-infected airway epithelial cell cultures. Neutrophil adherence was determined indirectly, using myeloperoxidase as a marker for adherent neutrophils in an assay system described here. Airway epithelial cell cultures (both primary human tracheal epithelial cells, and two permanent cell lines, A549 and BEAS-2B) were grown in 96-well tissue culture plates and infected with human parainfluenza virus type 2. Infected airway epithelial cell cultures supported significantly enhanced levels of neutrophil adherence (up to 50-75% of neutrophils added to the wells) compared to uninfected control cultures. Moreover, this adherence occurred in a virus dose-dependent fashion, with increasing levels of adherence noted at increasing viral multiplicities of infection. The assay system described allows the detection of small numbers of adherent neutrophils (as few as 1000 neutrophils) in a 96-well format.

Respiratory virus infection and airway hyperreactivity in children

A significant portion of infants and young children develop acute lower airway obstruction (wheezing) accompanying viral lower respiratory infections. These wheezing‐associated respiratory illnesses, particularly bronchiolitis, are often associated with evidence of prolonged airway obstruction and airway hyperreactivity following the initial infection. The factors that determine the type of clinical infection that initially develops and long‐term respiratory consequences of this infection are complex, including the genetic background of the child, the age at the time of infection, the respiratory virus involved, and environmental factors such as exposure to cigarette smoke. Several animal models have been developed to describe the pathophysiology of viral respiratory infection and have helped to define several potential mechanisms for the resulting airway obstruction. Data from these studies and studies in humans emphasize the importance of the immune system and of neural modulation of airway smooth muscle tone in determining the outcome of respiratory virus infection. These observations also help define the pivotal role of the airway epithelium in the regulation of airway smooth muscle tone and the host inflammatory response in the airway. Consequently, the interplay of host and environmental factors, as well as the relative importance of the different mechanisms potentially active in airway obstruction, determine the acute clinical outcome of infection for an individual child and the propensity of that child to develop recurrent episodes of wheezing (asthma).

IL-12p40 and IL-18 Modulate Inflammatory and Immune Responses to Respiratory Syncytial Virus Infection

Respiratory syncytial virus-induced bronchiolitis has been linked to the development of allergy and atopic asthma. IL-12 and possibly IL-18 are central mediators orchestrating Th1 and/or Th2 immune responses to infection. To determine a possible role for IL-12 in regulating the immune response to acute respiratory syncytial virus infection, IL-12p40 gene-targeted (IL-12p40−/−) and wild-type mice were intratracheally infected with respiratory syncytial virus, and lung inflammatory and immune responses were assessed. Lung inflammation and mucus production were increased in the airways of IL-12p40−/− mice as compared with those of wild-type mice, concurrent with increased levels of the Th2 effector cytokines IL-5 and IL-13. Respiratory syncytial virus clearance and levels of Th1 effector cytokine IFN-γ were not altered. Interestingly, IL-18, another mediator of IFN-γ production, was significantly increased in the lungs of IL-12p40−/− mice early during the course of infection. Abrogation of IL-18-mediated signaling in IL-12p40−/− mice further enhanced Th2 immune response and mucus production in the airways during respiratory syncytial virus infection but failed to modulate IFN-γ production or viral clearance. These findings implicate a role for IL-12 and IL-18 in modulating respiratory syncytial virus-induced airway inflammation distinct from that of viral clearance.