Antigen-induced Stimulation of Glucosamine Incorporation by Guinea Pig Peritoneal Macrophages in Delayed Hypersensitivity

The interaction between sensitized lymphocytes and specific antigen occurring in delayed hypersensitivity causes bystander macrophages to undergo a variety of light-microscopic, ultrastructural, and biochemical changes, which are reflected in alterations in cell movement and intercellular contacts. Since such alterations involve functions of the cell periphery, we postulated that metabolic changes in this polysaccharide-rich zone would accompany the expression of delayed hypersensitivity. We here demonstrate that the incorporation of radioactive glucosamine by peritoneal macrophages into TCA-precipitable, membrane-associated material is regularly enhanced when these are cultured in the presence of specific antigen and nonadherent cells (lymphocytes) primed for delayed hypersensitivity. Lymphocytes from unsensitized animals, or from animals immunized so as to form antibody but not delayed hypersensitivity, do not stimulate such incorporation. Antigen-induced glucosamine incorporation is maximal at 2 or 3 days of culture and is not observed earlier; it may be elicited with as little as 0.1 microg/ml PPD, and affords an exceedingly reproducible and sensitive index of delayed hypersensitivity. Radioautographic studies indicate that nearly all plastic adherent cells (90% macrophages) incorporate glucosamine and that grains are concentrated in the regions of the perinuclear zone and cell membrane. Subcellular fractionation indicates that nearly 30% of counts and the highest specific activity are associated with the membrane-rich microsomal fraction; the microsomal distribution of counts increases in both absolute and relative terms when macrophages are cultured in the presence of specific antigen and sensitized lymphocytes. Taken together, these data indicate that a sizable fraction of incorporated glucosamine is localized to the vicinity of the cell periphery but lack sufficient resolution to determine whether this material is associated with the cell membrane itself or with the extramembranous cell coat. This last possibility is of particular interest since we have previously shown that macrophage cell coat material is lost or altered as a consequence of an interaction between sensitized lymphocytes and specific antigen.