Daniel L. Luchtel

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.

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.

Bisulfite induced lipid oxidation.

Bisulfite (0.5 to 10 millimolar) initiates oxidation of lipids in aqueous emulsions of corn oil in a dose-dependent manner as measured by reaction with thiobarbituric acid. The reaction is effectively quenched by the antioxidant, 2,6-dit-butyl-4-hydroxymethyl phenol as well as by Mn+2 (10(-5)-10(-3)M). The latter observation is in disagreement with that reported by others and suggests that in case of lipids an alternative mechanism exists for the initiation of reactive species.

Ultrastructure of the lung of the rattlesnake, Crotalus viridis oreganus

Scanning and transmission electron microscopic observations were made on the rattlesnake lung, which has the form of a cigar‐shaped bag enclosing a large axial air chamber. The lungs were fixed by tracheal instillation of fixative to preserve the structural features of inflated lungs. An open tracheal groove along the ventral aspect of the lung is the only structural “airway” present. The wall of the lung has two histologically distinct regions: anteriorly, a respiratory portion, where up to three generations of septa subdivide the wall into cup‐shaped gas‐exchange chambers, termed faveoli; and posteriorly, a simple, thin‐walled saccular portion. The epithelium lining the internal surface of the lung is composed of several cell types: (1) ciliated cells; (2) type I pneumonocytes; (3) type II pneumonocytes, secretory cells characterized by the presence of lamellar bodies; and (4) serous epithelial cells, secretory cells characterized by the presence of homogeneous, densely staining secretory granules. However, the distinctiveness of the secretory cell types in the snake lung is blurred because intermediate‐appearing cells have both the lamellar body and homogenous type of secretory granule. The nonepithelial components of the pulmonary wall and septa consist of blood vessels and lymphatics, smooth muscle cells and fibroblasts, embedded in a matrix of extracellular connective tissue fibers. Tubular myelin figures were observed in the faveolar lining layer.

Histological Methods to Determine Blood Flow Distribution with Fluorescent Microspheres

We evaluated several histological methods and determined their advantages and disadvantages for histological studies of tissues and organs perfused with fluorescent microspheres. Microspheres retained their fluorescence in 7-10 microm serial sections with a change in the antimedium from toluene when samples were fixed in formalin and embedded in paraffin. Several antimedia allowed both wax infiltration of tissue and preservation of microsphere fluorescence. Histoclear II was the best substitute for toluene. When samples were fixed in formalin and embedded in glycol methacrylate, thinner (3-5 microm) sections provided greater histological detail but had fewer microspheres per section. Air dried lung tissue followed by Vibratome sectioning provided thick sections (100 microm) that facilitated rapid survey of large volumes of tissue for microspheres but limited histological detail, and the air drying procedure was restricted to lung tissue. Samples fixed in formalin followed by Vibratome sectioning of unembedded tissue provided better histological detail of lung tissue and was also useful for other organs. These sections were more difficult to handle and to mount on slides compared to air dried tissue, whereas fixed tissue embedded in gelatin provided better tissue support for Vibratome sectioning. Rapid freezing followed by cryo-microtome sectioning resulted in frozen sections that were relatively difficult to handle compared to embedded or unembedded tissue; they also deteriorated relatively rapidly with time. Paraffin sections were stained with hematoxylin and eosin or with aqueous methyl green, although tissue autofluorescence by itself was usually sufficient to identify histological features. Methacrylate sections quenched tissue autofluorescence, and Lees stain or Richardsons stain were used for staining sections. Toluene based mountants such as Cytoseal quenched fluorescence, particularly the red fluorescent microspheres. Aqueous based mountants such as Aquamount, Crystal/Mount, Fluoromount-G were substituted, although such preparations were not as permanent as Cytoseal mounted coverglasses and tended to cause fading of stained sections.

Gonadal development and sex determination in pulmonate molluscs

SummaryA cytological study has been made, at the light- and electron-microscope levels, of gonadal development in Arion circumscriptus. The gonad appears to be ectodermal in origin and is composed of two cell lines—germinal and non-germinal. These two lineages are established in the embryo. The germinal line is first represented by primordial germ cells while the non-germinal line is first represented by auxiliary cells. The primordial germ cells differentiate into spermatogonia and oogonia, the auxiliary cells into Sertoli cells and follicular cells. Spermatogonia and oogonia differentiate about the time of hatching. All subsequent germ cells appear to differentiate from these initial populations of spermatogonia and oogonia. The concept of a germinal epithelium, as a cell layer in which germ cells differentiate from indeterminate germ cells throughout the life cycle, is not supported by this study.A model for sex determination of the germ cells is proposed based on the hypothesis that sexually non-determined primordial germ cells are distributed into two physiologically isolated compartments (a male medullar compartment and a female cortical compartment). The spermatogonia and oogonia then differentiate in two different microenvironments.

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.

Ozone can increase the expression of intercellular adhesion molecule-1 and the synthesis of cytokines by human nasal epithelial cells

AbstractOzone is a highly toxic and ubiquitous air pollutant that induces epithelial cell injury and airway inflammation. This study examined the effects of sublethal, near ambient ozone concentrations on the expression of intercellular adhesion molecule-1 (ICAM-1, CD54) and the synthesis of certain cytokines by human nasal epithelial cells (HNE). The cytokines studied were interleukin-lα (IL-1α), interleukin-6 (IL-6), interleukin-8 (IL-8), and tumor necrosis factor-α (TNFα). The HNE were cultured and exposed to ozone in an environment that simulated that of the human airway. Cells were grown and exposed on microporous filters and were assayed for ICAM-1 expression in an adherent state with a laser cytometer. Dual-wavelength detection using a fltiorescein-conjugated antibody to detect receptor expression and propidium iodide to count cell nuclei allowed measurement of ICAM-1 expression on individual cells, expressed as fluorescence per cell. The IL-lα, IL-6, IL-8, and TNFα were measured in HNE culture sup...

Abnormal neurulation induced by 7-hydroxy-2-acetylaminofluorene and acetaminophen: Evidence for catechol metabolites as proximate dysmorphogens

Direct additions to culture media of either acetaminophen (APAP) or 7-hydroxy-2-acetylaminofluorene (7-OH-AAF) resulted in abnormal closure of the anterior neuropores of cultured rat embryos in the absence of an exogenous bioactivation system. Concentrations required to produce a 50% incidence of the defect were approximately 500 and 250 microM for APAP and 7-OH-AAF, respectively. Losses of viability were not evident at these concentrations but 7-OH-AAF elicited a somewhat greater effect on growth parameters and generalized embryotoxicity. Transplacental induction with 3-methylcholanthrene (MC) of P450IA1 in subsequently cultured rat embryos did not detectably alter the capacity of APAP or 7-OH-AAF to effect embryotoxicity or neuropore closure. However, additions to the culture medium of exogenous hepatic bioactivating systems (S9) from MC-induced vs phenobarbital (PB)-induced adult rats produced profoundly different effects on neuropore closure. Coincubation with S9 from MC-induced rats reduced the incidence of 7-OH-AAF-elicited abnormal neuropores from 45 to 19%, whereas coincubation with S9 from PB-induced rats increased the incidence to 77%. Coincubation with MC-induced S9 produced no statistically significant effect on APAP-elicited neuropore abnormalities but, with PB-induced S9, resulted in a significant increase from 60 to 86%. Additions of 3-OH-APAP (0.1-0.2 mM) but not N-acetyl-p-benzoquinoneimine (NAPQI, 0.1-0.5 mM) to the culture medium elicited the typical neuropore abnormality. Experiments with APAP and 7-OH-AAF as substrates demonstrated that embryonic enzymes catalyzed their conversion to the corresponding catechols. Considered together, the results provided evidence that embryonic conversion of APAP or 7-OH-AAF to the corresponding catechol metabolites may be instrumental in effecting the abnormal anterior neuropore closure observed after exposure of embryos to the respective parent compounds.