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Successful long-term culture of murine thymic macrophages was achieved by plating adherent thymic cells, in the presence of L cell-conditioned medium, on dishes coated with an extracellular matrix. Adherent thymic cells in normal conditions of in-vitro culture do not proliferate. Those maintained on plastic tissue-culture dishes, and exposed to L cell-conditioned medium, proliferate slowly to a limited degree and form very small colonies. In contrast, when cultured in dishes coated with an extracellular matrix formed by corneal endothelial cells, in the presence of L cell-conditioned medium, adherent thymic cells proliferate rapidly and after 12-21 days in culture form large colonies (about 3-5 mm in diameter). The proliferating cells were identified to be mononuclear phagocytes by their morphological appearance, their ability to ingest both bacteria and antibody-coated erythrocytes and by their nonspecific esterase activity. These cells were also shown to exhibit cell surface antigens that are characteristic of differentiated macrophages, e.g. Fc receptors and the specific macrophage cell surface marker F4/80. A high percentage of these cultured cells were found to bear I-A antigens. The adherent thymic mononuclear phagocytes could be trypsinized and passaged while maintaining both their ability to proliferate and their specific macrophage characteristics for a period of 70 days. Thus, monocyte-macrophage stem cells ae present in the thymus, and under appropriate in-vitro conditions, can be made to proliferate and mature to I-A-bearing macrophages.

Separate and independent phagocytic recognition sites have been identified on mouse peritoneal macrophages through the use of xenogeneic antimacrophage serum (AMS) and allogeneic anti H-2 antisera. Anti H-2d and anti H-2b antisera inhibit the binding and ingestion of opsonized erythrocytes (EA) by macrophages bearing H-2 haplotypes d and b, respectively. AMS and its F(ab')2 and Fab fragments inhibit the binding and ingestion of EA, and the ingestion but not the binding of 125I-labelled Shigella by macrophages. Neither antiserum inhibited the binding or ingestion of latex particles by macrophages. The results suggest that particulate binding to macrophages can be inhibited by two different mechanisms: a non-specific one where antibody bound to certain cell-surface antigens can mediate either directly or indirectly, and a specific interaction with Fc receptors. The possible mechanisms of non-specific antibody mediated phagocytic inhibition are discussed.

Mast cell differentiation was generated in the following three experimental situations: (i) infection of mice with Schistosoma Mansoni or with Nippostrongylus brasiliensis and growth of the lymph node cells in the presence of the corresponding helminth antigen; (ii) immunization with horse serum and growth of blood and lymph node cells in the presence of the horse serum; (iii) exposure of T-cell-depleted suspensions of lymph node cells from unimmunized mice to T-cell factor (TCF) released into medium of the young cultures of (i) and (ii). This differentiation was also obtained when lymph node cells from athymic nude mice were exposed to TCF. The cell suspensions were plated on X-irradiated fibroblast monolayers prepared from embryonic mouse skin. Screening of the suspensions before plating on the fibroblasts in culture revealed no young forms of mast cells, and none were present in culture of nude mice lymph node cells maintained without TCF. Primordial appearance of metachromatic granules generally in the golgi zone was first seen in many 'large lymphoid cells' as early as 18 hr after plating. This was followed by increase in the cytoplasm volume, increase in granule number and mitosis, ending at 10-18 days with homogeneous populations of mature mast cells. When the mesenteric lymph node cells from mice infected with the helminths were grown in the absence of fibroblasts but in the presence of the antigen, homogeneous populations of cells with extended cytoplasm, filled with unstained vacuoles developed during days 7-13. These cells did not contain histamine (or at most 0.2 microgram per 10(6) vacuolated cells). When these cells were plated on fibroblast monolayers clear granule formation in all the vacuoles was seen 2 days later. It increased progressively in size and staining intensity, until the vacuoles transformed into typical mast cell granules. By the fourth day the vacuolated cells attained the typical mast cell morphology and the histamine content greatly increased (from 0.12 microgram per 10(6) vacuolated cells to 3.02 micrograms per 10(6) mast cells). These mast cells were readily degranulated by monoclonal anti-DNP-BSA IgE, and the antigen, releasing 90% of the histamine. The study shows that mucosal mast cells formation from 'large lymphoid-like' cells present in the blood and in the lymph, is stimulated by TCF. The condensation of the metachromatic material and histamine synthesis depends on other cells, presumably fibroblasts which comprise the principal cell in the embryonic skin monolayers. The mechanism of the fibroblast influence is not yet known.

The present report examines the behaviour of a slowly proliferating pure population of thymus-derived macrophages in long-term culture, regarding their ability to secrete interleukin-1 (IL-1) and prostaglandin E2 (PGE2), to stimulate a mixed leucocyte reaction (MLR) and to lyse tumour cells in vitro. Following stimulation with LPS, high levels of IL-1 were released to the medium. IL-1 release was significantly augmented by the addition of indomethacin during stimulation. Thymus-derived macrophages constitutively secreted significant levels of PGE2. These cells served as excellent stimulators in a one-way MLR, substantiating the claim that pure populations of macrophages effectively stimulate an allogeneic response in vitro. Thymus-derived macrophages showed tumoricidal activity following activation with either high concentrations of LPS or suboptimal concentrations of LPS and T-cell lymphokine. These findings portray a close interrelationship and reciprocal regulation between thymus-derived macrophages and T lymphocytes.

Macrophages are cytotoxic to chicken embryonic fibroblasts without either previous activation or lymphocyte assistance. This cytotoxic activity (xenolysis) is expressed by non-activated macrophages from athymic mice as well as by pure macrophage populations. Neither macrophage lysate nor supernatants of macrophages cultivated with fibroblasts cause xenolysis. Unlike macrophage tumoricidal activity, killing of xenogeneic cells is not dependent on specific serum factors and is expressed by macrophages from a lipopolysaccharide (LPS) unresponsive strain (C3H/HeJ). Xenolysis is expressed also by trypsin-treated macrophages and by macrophages from 5-day-old cultures. Killing of chicken fibroblasts by macrophages is not affected by hydrocortisone (100 micrograms/ml) gold salt (1 mg/ml) and colchicine (100 micrograms/ml). On the other hand, cytochalasin B (10 micrograms/ml) completely abolishes the killing, probably by interfering with macrophage mobility and extension of filopodia toward the targets. It is suggested that the xenolytic activity of macrophages represents a primitive trait of phagocytes which assists the body in defence against multicellular parasites.

Artificial binding of mouse peritoneal macrophages to syngeneic embryonic fibroblasts by means of rabbit anti-mouse anti-macrophage serum or Con-canavalin A inhibited the proliferation of syngeneic target cells without destroying them. It is suggested that an intimate membranal contact between macrophages and target cells triggered cytostasis, whereas cytotoxicity requires an additional step of foreign recognition.

Exposure of thioglycollate-elicited murine peritoneal macrophages to wheat germ agglutinin (WGA) increased markedly the uptake of six different bacteria, which have surface receptors for the lectin. Uptake of Staphylococcus aureus H was higher by 3-5-fold, of S. aureus 52A2 by 1.8-fold, of S. aureus 52A5 by 1.7-fold, of S. albus by 2.3-fold, of Shigella flexneri by 6-fold and of Micrococcus luteus by 6.5-fold. Klebsiella pneumoniae, devoid of receptors for WGA, was not phagocytosed following pretreatment of macrophages with the lectin. Pretreatment of the bacteria with the lectin also resulted, in most cases, in an increase in phagocytosis. Interaction of WGA with the macrophages and with the bacteria, as well as the potentiation of phagocytosis, was abolished by tri-N-acetylchitotriose, a saccharide that binds specifically to WGA, but not by monosaccharides which do not interact with this lectin. With non-elicited macrophages, enhancement of phagocytosis by WGA was less pronounced, probably because of the higher number of lectin-binding sites (5-fold) on the elicited cells. Peanut agglutinin and soybean agglutinin, that bind to macrophages but not to the bacteria studied, lack the ability to potentiate phagocytosis. Macrophage surface sugars thus appear to play an important role in phagocytosis by serving as receptors for lectins that form bridges between the macrophages and the microorganisms.