Coralie Baker

Cytokines and eosinophil-derived cationic proteins upregulate intercellular adhesion molecule-1 on human nasal epithelial cells

BACKGROUND Allergic and nonallergic rhinitis with eosinophilia syndrome are characterized by tissue eosinophilia and nasal mucosal injury. Recently, it has been shown that the adherence of eosinophils and other leukocytes to epithelial cells is mediated by intercellular adhesion molecule-1 (ICAM-1) and related adherence-promoting glycoproteins. METHODS In this study we examined the constitutive expression of ICAM-1 on human nasal epithelial cells (HNECs), and the effects of interferon-gamma, tumor necrosis factor-gamma eosinophil major basic protein, and eosinophil cationic protein on the regulation of ICAM-1 expression on these cells. Similar studies were performed with A549 pneumocytes as comparative epithelial cells. RESULTS Constitutive expression of ICAM-1 was significantly higher on cultured HNECs than on A549 cells, although nasal epithelial cells in tissue specimens did not demonstrate detectable levels of ICAM-1. This spontaneous expression of ICAM-1 on cultured HNECs may explain the unique susceptibility of the nasal mucosa to rhinovirus infection, because ICAM-1 is the epithelial cell receptor for most rhinoviruses. Physiologic concentrations of major basic protein and eosinophil cationic protein stimulated significant upregulation of ICAM-1 on HNECs, which was comparable to that produced by interferon-gamma and tumor necrosis factor-alpha. In contrast, these eosinophil constituents did not stimulate ICAM-1 upregulation on A549 alveolar epithelial cells, although A549 cells did respond to interferon-gamma and tumor necrosis factor-alpha. CONCLUSION The observation that eosinophil products upregulate ICAM-1 on HNECs suggests a positive feedback mechanism, in which the products released from migrating eosinophils might promote additional HNEC-leukocyte adherence by enhancing interactions between leukocyte beta 2 integrins (CD11/18) and nasal epithelial ICAM-1.

Synthesis of interleukin-1α, interleukin-6, and interleukin-8 by cultured human nasal epithelial cells

Nasal epithelium forms the initial barrier between the environment and the respiratory system and may be a potential source of proinflammatory interleukins, which contribute to the pathophysiology of allergic and nonallergic rhinitis. To explore this possibility, epithelium and cultured human nasal epithelial cells from nasal turbinates of patients undergoing surgery for treatment of upper airway obstruction were examined for the spontaneous expression of interleukin (IL)-1 alpha, IL-1 beta, IL-6, and IL-8. Human nasal epithelial cell lysates and culture supernatants were assayed by two-site ELISAs specific for IL-1 alpha, IL-1 beta, IL-6, or IL-8. Maximum concentrations of these cytokines in supernatants ranged from approximately 0.2 to 2 ng/ml for IL-1 alpha, 1.5 to 7 ng/ml for IL-6, and 100 to 3000 ng/ml for IL-8. IL-1 alpha was predominantly cell-associated, whereas most of the IL-8 and all of the IL-6 were detected in the supernatant. Little or no IL-1 beta was detected by ELISA in the supernatants or cell lysates. Whole tissue turbinates and isolated epithelium were also examined for IL-1 beta, IL-6, and IL-8 mRNA expression by Northern blot analysis. IL-6 and IL-8 mRNAs were detected, whereas IL-1 beta mRNA was not. Furthermore, IL-6 and IL-8 release from human nasal epithelial cell cultures was enhanced by addition to the cultures of lipopolysaccharide, and IL-6 release was inhibited by polymyxin B. Thus human nasal epithelium may be a major source of IL-1 alpha, IL-6, and IL-8 in allergic and nonallergic rhinitis. Production of those proinflammatory cytokines by epithelial cells of the nasal and sinus mucosa may contribute to the pathologic and clinical events that occur in these diseases.

Heart-Rate Variability in the Apolipoprotein E Knockout Transgenic Mouse Following Exposure to Seattle Particulate Matter

Epidemiological studies show that the elderly and/or people with preexisting cardiovascular disease (CVD) are more susceptible to the adverse effects of ambient air pollution. Heart-rate variability (HRV) measured through electrocardiogram (ECG) is a sensitive and effective tool for monitoring the adverse effects of particulate matter (PM). Common HRV parameters used include the standard deviation of the interval between normal beats (SDNN), square root of the mean of the squared differences between normal beats (rMSSD), and distinct high, low, and very low components of frequency. Aged apolipoprotein E knockout transgenic mice, a model of CVD, were implanted with miniaturized ECG telemetry devices and intranasally exposed to saline, 50 μg Seattle PM2.5 (PM having a mean aerodynamic diameter of ≤2.5 μm), or silica. They were monitored for a 1‐d baseline prior to and for 4 d following exposure. After an initial increase in both heart rate and activity in all groups, there was delayed bradycardia with no change in activity of the animals in the PM- and silica-exposed groups. In addition, with PM and silica exposure there was a decrease in HRV parameters, suggesting a decrease in parasympathetic tone, which may lead to cardiac arrhythmia and mortality. Seattle PM is a toxic species that modulates the autonomic nervous system in a mouse model of CVD. This work is supported by U.S. EPA grant R-827355-01-0 and NIEHS grant P30 ES07033.

Human Nasal Epithelium: Characterization and Effects of in Vitro Exposure to Sulfur Dioxide

Human nasal turbinate tissue from surgical specimens was dissected free of connective tissue, and primary epithelial cultures were established by explant techniques. Transmission electron microscopy revealed that cultured cells retained homogeneous cytoplasmic granules, tonofilaments, and desmosomes and formed a homogeneous monolayer. The epithelial cells stained positively with cytokeratin antibodies AE1, AE3, and 35BH11 but failed to stain with two other cytokeratin antibodies, AE2 and 34BE12. Staining was also positive with anti-desmoplakin I and II but negative with antivimentin (43BE8), anti-desmin, and anti-human factor VIII antibodies. Cultured cells were exposed to filtered air or sulfur dioxide at 1-5 ppm for 30-60 min. Although there was no increase in cell lysis as measured by chromium-51 release, SO2 exposure significantly inhibited [3H]leucine incorporation compared to air exposure. This effect was dependent on both SO2 concentration and exposure duration. Control experiments revealed that these SO2 effects were not caused by the [H+] load produced by SO2 exposure. Electron microscopy of cells exposed to air or SO2 did not show any significant morphological differences.

The effects of ozone exposure on lactate dehydrogenase release from human and primate respiratory epithelial cells

Ozone is the most persistent, wide-spread air pollutant in the United States. Over one half of the population of the US lives in cities or suburban areas which do not meet the National Ambient Air Quality Standard for ozone which is 0.12 ppm averaged over 1 h. Controlled laboratory exposures of human subjects have shown that ozone exposure produces decreased pulmonary function, hyperresponsiveness to inhaled methacholine, inspiratory pain, and airway inflammation as assessed by bronchoalveolar lavage. However, the cellular mechanisms responsible for such effects are incompletely known. The present study examined the effects of ozone exposure at 0.50 ppm for 3 h on three types of cultured respiratory epithelial cells; primary cultures of human nasal cells and primate bronchial cells, and the A549 type II pneumocyte-derived cell line. Cells were grown to confluent monolayers in plastic 6-well plates and then exposed to ozone or filtered air on a tilting platform over a heated water bath. Lactose dehydrogenase release was significantly increased following ozone exposure of all cell types; a 75% increase from human nasal cells (P = 0.0002), a 79% increase from primate bronchial cells (P = 0.003), and a 69% increase from A549 cells (P = 0.02). These data suggest that even brief ozone exposure causes membrane injury to cultured human respiratory epithelial cells.

RANTES Production by Cultured Primate Endometrial Epithelial Cells

PROBLEM: RANTES (regulated upon activation, normal T cell expressed and secreted), is a chemokine with monocyte, macrophage, T lymphocyte, and eosinophil attractant and activating activities. This mediator has been detected in the peritoneal fluid of patients with endometriosis and in cultures of stromal cells from human endometrial and endometriotic tissue. To determine if endometrial epithelial cells were also a potential source of this mediator, primate endometrial epithelial cells were cultured in vitro and the constitutive and stimulated production of RANTES in these cultures was measured.

Release of RANTES from nasal and bronchial epithelial cells

RANTES is a chemokine with eosinophil attractant and activating activities. This study was undertaken to determine whether primary cultures of human nasal and primate bronchial epithelial cells produce RANTES and the effect of various cytokines and dexamethasone on the release of this chemokine. Nasal epithelial cells from 32 patients (HNE) and bronchial epithelial cells from 17 Macaca nemestrina monkeys (PBE) were cultured in vitro for 24 to 72 h with LPS, TNF-α, IL-1β, IFN-γ and TNF-α combined with IFN-γ and/or dexamethasone at 10 to 1000 µg/ml. Culture supernatants were assayed for RANTES by ELISA. RANTES synthesis was measured by immunoprecipitation. HNE and PBE released modest constitutive amounts of RANTES (350 to 1000 pg/ml) which did not increase with time in culture. Release of RANTES was stimulated by all activators except LPS in a time-dependent manner, with the greatest synthesis induced by the combined addition of TNF-α and IFN-γ. The combination of these activators also increased RANTES synthesis as determined by immunoprecipitation. Dexamethasone at 100 and 1000 µg/ml produced significant inhibition of stimulated RANTES release. These data indicate that normal nasal and bronchial epithelial cells release RANTES which is upregulated by various cytokines and inhibited by dexamethasone. The enhanced release is due to stimulation of both synthesis and secretion. Production of RANTES by epithelial cells could contribute to the inflammation that characterizes the respiratory tract in asthma and rhinitis and downregulation of RANTES by glucocorticoids may be one mechanism of the therapeutic effect of these agents.