Use of a Sensitive Electro-optical Method to Quantify Superoxide Production from Single Pulmonary Alveolar Macrophages Exposed to Dusts in vitro or in vivo: Some Current Experimental and Model Results

"This laboratory bas developed a sensitive electro-optical method to quantify the initial rate (R) and total amount (MAX) of superoxide (02) produced by single pulmonary alveolar macrophages (PAM). The method uses a microscope-based TV system to visualize PAM in culture, and to video record the images during the time the cells produce 02. MAX and R are calculated from measurement of temporal changes in optical density in the images due to precipitated diformazan formed by the reaction of O2-, produced by each PAM, with nitroblue tetrazolium present in the culture medium. To date, values of Rand MAX, measured due to adherence of PAM to the dish, have been compared to values obtained when quartz, coal mine dust (CMD), and kaolin . were added to the medium (in vitro). Rand MAX have also been calculated for PAM lavaged from animals exposed to quartz and CMD in the WVU Inhalation Facility. Presently, experiments are being done using which will permit PAM to be restirnulated by different dusts to help establish a dose response relation~ p and a means to study the role of lung surfactant on modifying the acute toxicity of inbaled dusts. Ultimately, this methodology should provide useful insight into establishing the role of 02 in PAM dysfunction due to inhalation of dusts, and in lung diseases such as pneumoconioses. INTRODOCTIONPAM are free cells found in the lungs which protect the lungs by removing foreign debris and bacteria. This is accomplished, in part, by the process of phagocytosis, whereby foreign matter is internalized into vesicles known as primary phagosomes. Phagocytosis also involves the chemical breakdown of inhaled dusts and the killing of microbes. Detoxification is aided by the respiratory burst, which is a metabolic response of the cell to foreign substances, that results in the production of highly reactive oxidizing agents from the partial reduction of extracellular oxygen to superoxide (02) at the surface of the plasma membrane. Superoxide undergoes either spontaneous or enzyme catalyzed dismutation reactions to form hydrogen peroxide (H202) and subsequently hydroxyl radical (OH•) and singlet oxygen (02 t ). However, while these oxygen metabolites aid in the killing of microbes they also may destroy endogenous tissue. For example, 02 has been linked to the aging process and to many diseases including emphysema, diabetes, and cancer.'' Therefore, a better understanding of the production of 02 is extremely important since an abnonnally low production could result in damage by inhaled dusts and bacteria while an abnormally high production could result in direct damage to the lung tissue by the phagocytes. Inhalation of respirable sized mineral dusts, such as quartz (silica), coal mine dusts (CMD), and asbestos, results in various pulmonary disorders. PAM are thought to play an important role since evidence suggests that the first step in fibrogenesis is an interaction of dust particles with PAM. Generally, in vivo exposure of animals to mineral dusts results in increased respiratory burst activity, migratory patterns, phagocytic behavior, and secretory potential of PAM, and for this reason these have been implicated to be pivotal events in the pathogenesis of pulmonary diseases.1 In addition to stimulation of resident PAM, an influx of monocytes into the lung and/or production of new macrophages generally occurs in response to fibrogenic dust inhalation. Further, PAM may attract and stimulate fibroblasts, which normally synthesize proteins and collagen during rep~ of tissue, by secreting chemoattractants and enzymes. Thus, PAM may be involved in pulmonary disorders through failure or partial loss of their defensive capability, or indirectly, through release of other mediators."