Effects of In vitro Atmospheric Ammonia Exposure on Recovery Rate and Luminol-dependent Chemiluminescence of Bovine Neutrophils and Bronchoalveolar Macrophages.

Abstract

ABSTRACT. The effects of atmospheric ammonia, a major pollutant in animal confinement facilities, on bovine neutrophils and bronchoalveolar macrophages were evaluated in vitro. Ammonia exposure at concentrations 50, 100 and 200 ppm for one hour impaired recovery rates of neutrophils dose-dependently but enhanced their chemiluminescence activity per cell at lower concentrations (50 and 100 ppm). Macrophages were resistant to the exposure. Their recovery rates and chemiluminescence remained unaffected even at 200 ppm exposure. The present results suggest that ammonia exposure is unfavorable for bovine neutrophils in vitro, and probably in vivo also, in light of causing cell damage and triggering wider inflammatory responses.-KEY WORDS: ammonia exposure, bovine bronchoalveolar macrophage, bovine neutrophil.J. Vet. Med. Sci. 61(3): 279-281, 1999 monitored with a gas detector (Gastec model 801, Japan) before and after the cell exposure. Preliminary experiments have confirmed that ammonia concentration can be kept at each designed level at least for 2 hr at 37°C. Two ml of cell suspension were placed into a siliconcoated polypropylene V-bottom tube with a 25 mm diameter and a 50 mm depth in the chamber and exposed to each ammonia concentration for one hour at 37°C. After the exposure, pH values of the cell suspensions were measured with a pH meter (pHBOY-P2, Shin-Dengen, Japan). Ammonia concentrations dissolved in the suspension were estimated with a pH-ammonia concentration standard curve obtained preliminarily. Cell counting was made on each suspension and cell recovery rate at each concentration was calculated as a percentage of cell count ratio (Exposure/ Control). Recovered cells were centrifuged and resuspended in HBSS (pH 7.4) at a concentration of 1 × 10 6 /ml.The chemiluminescence assay of the recovered cells was performed as previously described [7]. Briefly, photon emission levels of the cells (2 × 10 5 cells) in response to phorbol myristate acetate (PMA: Sigma, 1.6 × 10 6 M at final concentration) were evaluated in the presence of luminol (Sigma, 0.5 mg/ml at final concentration) with a photon-counting luminescence analyzer (Berthold LUMAT LB9505C, Germany). The peak chemiluminescence (cpm) per cell at each ammonia exposure was calculated.Data were analyzed using analysis of variance and Student's t-test. A level of p<0.05 was regarded as significant. Table 1 shows recovery rates of neutrophils and macrophages exposed to ammonia at concentrations of 50, 100 and 200 ppm for one hour. Neutrophils were sensitive to ammonia exposure and their recovery rates decreased dose-dependently. In contrast, macrophages remained resistant to the exposure. The pH values and estimated ammonia concentrations (parentheses) in the cell suspensions were at pH8.4-8.6 (1-2 mg/dl), pH8.7-8.9 (2-3 mg/dl) and pH9.1-9.2 (5-6 mg/dl) at 50, 100 and 200

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