Bovine gut-associated lymphoid tissue--morphologic and functional studies. I. Isolation and characterization of leukocytes from the epithelium and lamina propria of bovine small intestine.

A successful technique for the isolation of highly pure suspensions of viable leukocytes from the small intestine of cattle is described. Procedures ranging from mechanical mincing to enzymatic digestion of tissues were compared. The most reliable and reproducible procedure was the sequential treatment of tissues with dithiothreitol (DTT), ethylenediaminetetraacetic acid (EDTA) in calcium-magnesium-free salt solutions, and collagenase. Two populations of mucosal leukocytes were obtained from the small intestine. One population was derived from within the epithelium (intraepithelial leukocytes, IEL), the second from within the lamina propria (lamina propria leukocytes, LPL). At least 2 X 10(6) viable leukocytes were obtainable from each square centimeter of the intestinal mucosa from either the epithelium or lamina propria. Erythrocyte rosetting and immunofluorescence characterization with conventional antisera and monoclonal antibodies (MAB) demonstrated that IEL were predominantly T cells (60%), with relatively few B cells present (10%), while LPL contained relatively high numbers of B cells (28%) and a reasonable percentage of T cells (45%). Both cell populations proliferate in response to stimulation with T and B cell mitogens. Addition of the thiol compound, 2-mercaptoethanol (2-ME) strongly augmented the mitogenic response of both cell isolates. Human recombinant interleukin-2 (hr-IL-2) in the presence or absence of additional stimuli was found to be able to induce the proliferation of both cell types. These results demonstrate that functional leukocytes can be isolated from the small intestine of cattle, and that they can maintain their responsiveness to both T and B cell mitogens and to exogenous cloned IL-2.