The effect of gamma-irradiation on the adherent capacity and iron metabolism of alveolar macrophages in mice and rats.

The effect of external gamma-irradiation on the survival of alveolar macrophages (AM) and on the extracellular release of 59Fe from AM-ingested [59Fe]iron hydroxide colloid was investigated in vitro using cells from C3H mice and Wistar rats. 59Fe release from mouse AM was enhanced by irradiation in a dose-dependent fashion, but irradiation up to 112 Gy did not affect the release from rat AM. Cell survival of AM, measured by direct counting of cell nuclei of adherent AM or vital staining with crystal violet, decreased dose dependently in mice and rats, but mouse AM were more radiosensitive than rat AM.


Introduction
Alveolar macrophages (AM) are thought to be important scavengers in the lung due to their ability to phagocytize, transport, and digest inhaled particles, after which they may receive a relatively large radiation dose directly from inhaled radioactive particles (1). Ifphysiological functions ofAM are affected by irradiation, the movement and metabolism ofradioactive particles might be altered. However, little is known about the effect of irradiation on AM (2)(3)(4)(5). In the present study, the effect of -y-irradiation on AM from C3H mice and Wistar rats was investigated, and it was found that radiosensitivity of AM is considerably different between the species.
Address reprint requests to Y. Kubota, Division of Comparative Radiotoxicology, National Institute of Radiological Sciences, Anagawa4-9-1, Chiba 260, Japan. suspended in Eagle MEM and irradiated with a 137Cs unit at a dose rate of 12.5 Gy per minute. Immediately after irradiation, we plated the cells in 96-well microtest plates and allowed them to adhere for 2 hr. Adherent cells (AM) were washed with PBS to remove nonadherent cells and cultured in fresh medium up to 3 days. In some experiments, a chelating agent, Cadiethylenetriamine pentaacetic acid (DTPA), and macrophage activating substances (lipopolysaccharide and interferon -y) were added to the medium. At 24, 48, and 72 hr after irradiation, we determined the rate of59Fe release by measuring the activity of59Fe in the supernatant and in the adherent cells with an automatic well type gamma counter.
Effect of y-Irradiation on Survival of Alveolar Macrophages AM were prepared, irradiated, and cultured by the same methods as described above, except that AM were not loaded with 59Fe-colloid. The survival of AM was determined by two different methods. In the first method, we determined the survival (more exactly, the number of adherent AM after washing with PBS at the end of culture) by counting the number of cell nuclei with a hemocytometer after the cell membranes were solubilized by adding 1% Zapoglobin-Il in PBS. In the second method, we determined the survival by using the vital staining method with crystal violet, where AM adhering to the plates after washing with PBS were stained with 0.1% crystal violet, and absorbance ofeach sample was measured at a wavelength of540 nm by a spectrophotometer (6).

Results and Discussion
The effect ofirradiation on the release of59Fe from mouse and rat AM loaded with [59Fe]iron hydroxide colloid was investigated. As shown in Table 1, 59Fe release from mouse AM was enhanced dose dependently, but rat AM was not affected by irradiation up to 112 Gy. We hypothesize that enhancement of 59Fe release from mouse AM was due to the impairment of cellular integrity because nuclear pyknosis, fragmentation, and autolysis were observed microscopically in irradiated mouse AM. Therefore, the effect ofCa-DTPA, interferon y, lipopolysaccharide, and irradiationon 5Fe release was studied using only rat AM. The combination of Ca-DTPA, interferon 'y, and lipopolysaccharide remarkably increased the release of 59Fe from AM, but 'yirradiation showed no effect ( Table 2). Next we investigated the effect ofirradiation on the survival of AM from both species. The survival of mouse AM estimated by the direct cell nuclei counting method was decreased to 68, 52, and 42% of nonirradiated controls by 28, 56, and 112 Gy of irradiation, respectively, at 24 hr after irradiation.
On the other hand, the survival of rat AM did not decrease. The estimation of the survival of AM by vital staining with crystal violet was more sensitive than that by direct cell nuclei counting. Also in this assay system, mouse AM were more radiosensitive than rat AM at every timepoint and atall doses examined (Fig. 1). The survival curve ofAM wasalsodifferentbetweenthese species; it was exponential with dose in mice, but not exponential in rats.
In conclusion, we found that AM from Wistar rats were relatively more radioresistant than AM from C3H mice with respect to survival. b"Fe activity released to supenant from rat AM during 48 hr after irradiation expressed as percentage of whole 59Fe activity.