A monoclonal antibody (BM8) raised in the rat against cultured mouse bone marrow-derived macrophages reacted only with macrophages and not with granulocytes, mast cells, platelets, lymphocytes, fibroblasts, and endothelial cells. BM8 did not detect blood monocytes. In cultured bone-marrow cells, expression of BM8-antigen was found on a few macrophages after one day of culture and reached its maximum level with 80% positive macrophages after 7-10 days of culture. The antibody BM8 belonged to the IgG2a subclass, was non-cytotoxic and directed against a 125 kD membrane antigen. In cryostat sections of normal mouse tissues (spleen, lymph node, thymus, liver, skin) BM8 detected tissue-fixed macrophages and Langerhans cells in the skin. In spleen and lymph node, BM8 reacted with macrophages in the red pulp and in the medullary cords, respectively, but not with heavily phagocytosing marginal-zone macrophages, as revealed by in-vivo phagocytosis of colloidal carbon. In granulomata induced by complete Freund's adjuvant, BM8 detected inflammatory macrophages but not epithelioid cells. Thus, antibody BM8 detected a differentiation antigen expressed only on mature, tissue-fixed macrophages.

It has been described before that only certain types of macrophages are capable to respond to lymphokines and that only certain macrophage phenotypes were able to migrate and to respond to migration inhibitory factors (MIF). With respect to the dissociation of MIF activities from a series of other biological activities, and with regard to the phenotype-associated response of macrophages to MIF it was asked: What are the characteristics of the MIF-responsive macrophage phenotype and what are the functional changes induced by MIF on macrophages in addition to inhibition of random migration? Bone marrow-derived macrophages on day 6 of culture were separated by hypotonic Percoll density gradient centrifugation into three distinct bands and analyzed for a variety of functions. It was found that migrating and MIF-responsive macrophages accumulate at a certain density. These macrophages were further characterized by monoclonal antibodies generated against murine macrophage phenotypes. One marker was found to be preferentially expressed by MIF-responsive macrophages. In order to study the inducibility of MIF responsiveness, bone marrow-derived macrophages on day 16 of culture which were poorly migrating and did not respond to MIF were induced to proliferate by the addition of L cell-conditioned medium. After proliferation had subsided, MIF sensitivity was restored. The effects of MIFs other than migration inhibition, on a number of functions which had been mapped within the cell cycle, were investigated. It was found that MIF acts anti-proliferative on "young", cycling macrophages. Non-cycling, mature macrophages were shifted to a state characterized by a decreased expression of transglutaminase and plasminogen activator and an increase of certain phenotypic surface markers. It is concluded that MIFs are differentiation-inducing signals, acting on the generation of macrophages from precursors but also in the recruitment of terminally differentiated macrophages to "inflammatory" type of macrophages which are functional in the induction of immune responses.

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Mouse bone marrow cells differentiate in culture in the presence of L cell-conditioned medium to macrophages (M phi). Proliferation, release of plasminogen activator, expression of transglutaminase, random motility and response to a standard preparation of purified M phi migration inhibitory factor (MIF) was recorded daily up to 14 days. After an initial phase of proliferation, precursor cells differentiated into M phi. In the course of maturation, plasminogen activator production was transiently expressed between day 4 and 12; beginning on day 5 the cells expressed intracellular transglutaminase. Random motility of cells was high at the beginning of culture but steadily declined thereafter. The response to MIF was only expressed between day 5 and 8. However, it was possible to induce MIF responsiveness in mature, unresponsive M phi by the addition of L cell-conditioned medium. To characterize the MIF-responsive M phi type further, bone marrow-derived M phi at day 6 of culture were separated on a hypotonic Percoll gradient into three distinct cell bands. While all densities of M phi displayed random migration, only cells with a density between 1.060 and 1.065 were responsive to MIF. We conclude that the response of M phi to MIF is a phenotypic trait transiently expressed in the course of maturation.