A study was performed to determine whether equine peritoneal macrophages produce interleukin 6 (IL-6) in vitro in response to endotoxin. Peritoneal fluid was collected from 14 clinically normal adult horses and was used as the source of peritoneal macrophages. Macrophages from each horse were isolated and cultured separately in vitro in the absence or presence of various concentrations (0.5, 5, or 500 ng/ml) of endotoxin (lipopolysaccharide from Escherichia coli 055:B5). Culture medium supernatants were collected after 3, 6, 12, and 24 hours' incubation and were frozen at - 70 C until assayed for IL-6 activity. Supernatant IL-6 activity was determined by use of a modified colorimetric assay and the murine hybridoma cell line B13.29 clone B.9, which is dependent on IL-6 for survival. Results indicated that equine peritoneal macrophages produce IL-6 in vitro and that supernatant medium IL-6 activity was significantly (P < 0.05) increased by exposure to endotoxin. Significant (P < 0.05) time and treatment effects on macrophage IL-6 production were apparent. The IL-6 activity peaked at 6 or 12 hours' incubation, then remained high through 24 hours' incubation, regardless of endotoxin exposure. Medium IL-6 activity during 3 and 6 hours' incubation was significantly (P < 0.05) greater in macrophages exposed to 5 or 500 ng of endotoxin/ml than in those exposed to 0.5 ng of endotoxin/ml; however peak IL-6 activity was similar among all endotoxin concentrations. Endotoxin concentration did not have an effect on medium IL-6 activity from macrophages exposed to endotoxin for 12 or 24 hours.

Addition of alanine and taurine blocked killing of lymphocytes caused by culture at 45 C. The optimal concentration for thermoprotection was achieved at 12.5 mM for L-alanine and 5 mM for taurine. Both D and L forms of alanine provided thermoprotection. The effect of these agents was not simply to increase osmolarity of the culture medium, because NaCl did not provide thermoprotection at comparable concentrations. Alanine and taurine were each tested at concentration of 50 mM for ability to block heat shock-induced killing and developmental retardation of 8- to 16-cell mouse embryos. Both agents enhanced embryo development after exposure to high temperature, though development remained less than that for embryos not exposed to high temperature. In one experiment, for example, 81% of embryos cultured at 38 C advanced in development during culture vs 0% at 42 C, 15% at 42 C with alanine, and 32% at 42 C with taurine. The beneficial effect of alanine at high temperature may have been partly attributable to effects independent of thermoprotection, because development of embryos cultured at 38 C was also improved by alanine.