Research Symposium: Basic Mechanisms and Human Health
Session 5. New Directions in Pulmonary Research
Cutaneous Application of Beryllium Salts and Oxide Particles Produces Beryllium-Specific Peripheral Sensitization in the C3H/HeOuJ Mice
Sally Tinkle, James Antonini, Jenny Roberts, Rebecca Salmen, Karyn Depree,
and Melanie Flint
Chronic Beryllium Disease is an occupational lung disease that begins as a cell-mediated immune response to beryllium. Because major improvements in respiratory protection did not decrease the rate of sensitization or disease over the last decade, and skin exposure to environmental chemicals is a well established route for immunologic sensitization, we hypothesized that skin exposure to beryllium could provide an alternative route for sensitization to this metal. Furthermore, recent evidence suggests that ultrafine beryllium particle concentration, not mass measurements, is the appropriate metric for relating exposure to risk of disease, and BeO particles are a common form of industrial exposure. We have tested fine particle penetration of human skin, and beryllium salt and beryllium oxide particle induction of peripheral sensitization in C3H/HeOuJ mice. We have demonstrated, by confocal microscopy, that 0.5 and 1 micron fluorescently-labelled dextran beads, in conjunction with flexing motion, as at the wrist, can penetrate stratum corneum and reach the epidermis. These data provided proof of concept for particle penetration into intact skin. To determine if epicutaneous beryllium causes peripheral sensitization, we applied BeSO4 solution or BeO particle suspension to the shaved backs of mice, and a single BeSO4 challenge on the ear. Forty-eight hours post-challenge, we measured by flow cytometric analysis of auricular lymph node cells, a significant increase in the B220 ratio and in the T cell activation marker, CD44. No change in surface marker expression was observed in mice sensitized with vehicle and challenged with BeSO4. Furthermore, we measured a concomitant decrease in CD62L on both CD4 and CD8 T lymphocytes, a cellular event also associated with T lymphocyte activation. To evaluate the antigen specificity of this response, we applied beryllium salts to the ears of mice and evaluated beryllium-induced lymphocyte proliferation (murine BeLPT) in vitro. We observed significant beryllium-induced auricular lymph node lymphocyte proliferation and peripheral blood mononuclear cell proliferation, with stimulation indices of ten and fifteen, respectively. In combination, these data provide evidence for beryllium salt and BeO particle induction of peripheral sensitization in mice and are consistent with development of an antigen-specific, cell-mediated immune response.
In Vitro Transformation of Phagocytized Beryllium Oxide Particles in the Murine J774A.1 Cell
G.A. Day1, A.B. Stefaniak2, M.D. Hoover3,
R.M. Dickerson4, E.J. Peterson1, N.A. Esmen5,
and R.C. Scripsick2
Beryllium (Be) metal, its oxide (BeO), and alloys are materials of industrial significance with recognized adverse effects on worker health. Currently, the degree of risk associated with exposure to these materials in the workplace is assessed through measurement of Be aerosol mass concentration, the control of which has proven ineffective at eliminating the occurrence of chronic beryllium disease (CBD). This might be due to intracellular change in BeO particles from crystalline to amorphous form as a potential mechanism by which dissolution occurs, thereby producing dissolved Be associated with the onset and progression of CBD. We examined changes in pre-characterized BeO particles engulfed by and retained within murine J774A.1 cells in vitro.
The physicochemical properties of commercially available BeO powder were analyzed by transmission electron microscopy (TEM) and x-ray powder diffraction (XRD). J774A.1 cells were dosed with BeO particles in vitro and incubated from 124 to 144 hours, followed by recovery of engulfed particles for post-characterization. Dissolved Be in recovered intracellular fluid was analyzed and quantified using inductively-coupled plasma atomic emission spectrometry (ICP-AES).
Pre-analytical TEM and XRD results showed a highly pure BeO powder consisting of diffuse clusters of primary particles measuring approximately 200 nm in diameter. Post-analytical TEM results did not show an observable change in morphology, chemical composition, or size. ICP-AES of the cell lysate indicated measurable levels of Be ranging from 3.2 to 27.4 parts per billion. These findings demonstrate the intracellular dissolution of BeO particles thereby showing that dissolved Be, concentrated within the cell, may serve as input to immunopathologic response in the host.
The Mouse Deep Lung is Exposed to Respirable Particles Administered by Pharyngeal Aspiration
A.F. Hubbs, G.V.S. Rao, M. Kashon, R. Salmen, L.A. Battelli, P. Willard,
J. Antonini, D.N. Weissman, and S. Tinkle
A pharyngeal aspiration technique has recently been described as a method for exposing the mouse lung to soluble antigens. This new technique overcomes the technical difficulty and potential trauma associated with intratracheal instillation methods in the mouse. In this study, we investigated the potential for the pharyngeal aspiration technique to expose the mouse deep lung to respirable particulates. Ten, 6-8 week old C3H/HeOujMMTV-, male mice were anesthetized with isoflurane using a bell jar. The mice were removed from the bell jar when anesthetized, placed on a dosing board with the mouth fully opened and the tongue gently held in full extension. A suspension of 1 micron blue fluorescent polystyrene amine-modified latex beads in a 50 microliter volume was pipetted onto the base of the tongue in the pharyngeal region. The tongue was maintained in fixed full extension until the mouse completed a minimum of two full breaths. Evaluations were made at 5 bead concentrations, representing a 100-fold range, with 2 animals per group. Anesthetic recovery occurred within one minute. Mice were necropsied four hours after exposure and no gross lesions were noted. Fluorescent beads were morphometrically quantified in frozen sections of lung. For each dose group, the percent of each microscopic field occupied by fluorescent beads directly and highly significantly correlated with number of beads administered (r2 = 0.99). Both the left and right lungs were exposed in all individuals, although the concentration of beads in the two sides varied in some mice. Beads were distributed in alveoli and alveolar ducts as free or phagocytized particles. We conclude that pharyngeal aspiration of particles is a simple, humane and effective technique that exposes the mouse deep lung to respirable particles over a wide range of exposure concentrations.
Beryllium-Induced Macrophage Apoptosis in Chronic Beryllium Disease
R.T. Sawyer, V.A. Fadok, L.A. Maier, L.A. Kittle, J.M. Routes,
and L.S. Newman
The link between metal-induced apoptosis and granulomatous inflammation in human disease pathogenesis is not established. The presence of TUNEL positive nuclei in chronic beryllium disease (CBD) pulmonary granulomas suggested the possibility that beryllium (Be) could induce apoptosis in CBD bronchoalveolar (BAL) cells. Apoptosis was measured in unstimulated and Be-stimulated BAL macrophages from CBD (n = 21) and Be-sensitized (BeS, n = 16) subjects. Be-stimulated CBD and BeS macrophages underwent cytoplasmic membrane blebbing, surface CD14 loss, and nuclear fragmentation. Nuclear fragmentation was blocked by the general caspase inhibitor BD-fmk. Apoptosis, CD14 loss and TNF-a production were not observed in unstimulated BAL cells. Be induced apoptosis of two mouse macrophage cell lines, only one of which produced Be-stimulated TNF-a. Be induced apoptosis of bone marrow derived macrophages from TNF-a (-/-) knockout mice. Thus, Be-induced macrophage apoptosis was caspase-mediated, TNF-a independent, and it occurred whether cells were derived from patients with granulomatous inflammation or not. Our data suggest that Be-induced macrophage apoptosis could contribute to the hostís continued re-exposure to Be. Beryllium-antigen induced macrophage apoptosis may represent a fundamental mechanism that separates antigen elimination and the resolution of inflammation, from antigen persistence and progression to chronic granulomatous inflammation.
(Supported by NIH grants ES06538 and GM60449)
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