Enzymatic digestion with pronase and DNAase was used to isolate Kupffer cells from mouse liver. The characteristics of these cells were found to be similar to those of peritoneal macrophages, except that in the initial suspension the percentage of Kupffer cells with Fc receptors was low, C receptors were absent and the ingestion of opsenized bacteria was very poor, because of the effect of pronase on the cell membrane. After 24 h incubation in vitro all these characteristics return. The in vitro and 1 h-pulse [(3)H]thymidine labeling of the Kupffer cells is low (0.8 and 1 percent, respectively) indicating that in essence these cells do not divide. It was also shown that the small percentage of in vitro labeled Kupffer cells was recently derived from the circulation. After an intravenous injection of zymosan the in vitro labeling index of the Kupffer cells increased 16-fold, but it was proven that these dividing cells were immature mononuclear phagocytes very recently recruited from the bone marrow. The labeling of Kupffer cells aider one or four injections of [(3)H]thymidine reached a peak of 10.4 percent at 48 h or 24.1 percent at 60 h, respectively, indicating that these cells are derived from labeled monocytes. Further evidence for this conclusion was obtained by the absence of an increase of labeled Kupffer cells during treatment with hydrocortisone, which causes a monocytopenia during which no circulating monocytes are available to migrate to the tissues. Labeling studies in animals X-irradiated with hind-limb shielding gave a Kupffer cell labeling index of 5-10 percent of the normal values, which confirms their bone marrow origin. A quantitative study on the production of labeled monocytes in the bone marrow and their transit through the circulation showed that in the normal steady state at least 56.4 percent of the monocytes leaving the circulation become Kupffer cells. Considering the Kupffer cells as kinetically homogeneous this gives a mean turnover time of the total population of Kupffer cells of 21 days.

During the last two decades, ample evidence has been obtained that osteoclasts, the multinucleated calcified-matrix resorbing giant cells of bone, which form by fusion of mononuclear precursor cells, are of hematogenous origin. The evidence stems from experiments done in parabionts of labeled animals (1), studies on osteopetrotic animals and humans (2-4), and quail-chick and mouse-quail transplantation experiments (5-7). Essentially, these in vivo studies have shown that osteoclasts derive from bone marrow or other hematopoietic tissues and have indicated mononuclear phagocytes as the most likely candidates for the precursor cells which fuse to form an osteoclast. However, in vitro evidence for the direct transformation of monocytes and/or tissue macrophages into bone-resorbing osteoclasts is still lacking, although it has long been known that cultured macrophages can form foreign-body giant cells in vitro by fusion (8-10).Several in vitro studies have dealt with the destruction of calcified bone matrix by mononuclear phagocytes; for this work, use was made of human peripheral blood monocytes (11, 12) or rodent macrophages (13) in combination with devitalized bone particles. Monocytes and macrophages were able to resorb mineral in a contactmediated fashion, but did not form cells with the morphological characteristics of osteoclasts (13). Recent investigations, however, point to the importance of interactions between bone-forming and -resorbing cells during osteoclastic bone resorption (14). This means that studies done on devitalized bone without viable bone-forming cells might be of limited value with respect to the formation of osteoclasts and osteoclastmediated bone resorption.We recently found (7) that early removal of the perichondrium-periosteum from embryonic mouse long-bone primordia prevents the formation of osteoclasts during organ culture of such bones. In mouse-quail transplantation studies, such stripped bone rudiments are invaded by quail osteoclasts, but mouse osteoclasts are not formed because the stripping procedure has removed the osteoclast precursor cells.In the present study, stripped live bone rudiments were used to assess the capacity of various populations of mononuclear phagocytes to form osteoclasts in vitro. Stripped bone rudiments were co-cultured with embryonic liver as well as with * Present address:

The main defenders of the respiratory organs against microorganisms and other foreign substances are the pulmonary macrophages. The majority of these cells, which belong to the mononuclear phagocyte system (1), are normally located in the alveolar spaces, where they phagocytize surfactants (2) and various substances introduced via the airways ; the remainder occur in the interstitial lung tissue (3) . The origin and kinetics of the pulmonary macrophages have been controversial, mostly because of differences in the interpretation of DNA-labeling characteristics in the absence of accurate quantitative information. Recently, a method developed to study the total pulmonary macrophage population by optimal lavage of the airways followed by enzyme digestion of lavaged lung tissue after removal of circulating monocytes from the pulmonary blood vessels enabled us to demonstrate that the great majority of the pulmonary macrophages of mice in the normal steady state derive from circulating monocytes originating in the bone marrow (4), and that local proliferation of mononuclear phagocytes does not play a significant role in the maintenance of the pulmonary macrophage population . ' The origin and kinetics of the increased number of pulmonary macrophages during acute inflammatory reactions are, however, still a matter of debate . On the basis of studies done of inflammation induced by various stimuli ranging from inert particles to pathogenic microorganisms and noxious gases in various animal models, both an influx of circulating monocytes and interstitial multiplication of macrophages or macrophage-precursor cells have been claimed to contribute to the pulmonary macrophage population under these conditions (5-9) .The present report concerns the macrophage kinetics during an inflammatory reaction in the lungs after the intravenous injection of heat-killed bacillus CalmetteGuerin (BCG).2 The kinetic patterns were studied by following the course and determining the DNA-labeling characteristics of both the circulating monocytes and the total macrophage population, i.e., the alveolar and interstitial macrophages. 2 Abbreviatons used in this paper. AML, alveolar-macrophage-like ; BCC, bacillus Calmette-Guerin ; C, complement ; HC, hydrocortisone acetate; NAML, non-alveolar-macrophage-like ; t DNA-synthesis time; ZN, Ziehl-Neelsen . J . Exp . MED .

The morphological and growth characteristics of human macrovascular endothelial cells (ECs) from venous and arterial umbilical cord vessels and microvascular ECs from foreskin were compared during cultivation. By means of time-lapse microcinematography and phase-contrast microscopy, differences in cell morphology and migratory activity between the different types of ECs were found. Growth characteristics were dependent on the type of EC, the nature of the substrates on which the ECs were grown and the presence of growth factors. For all types of ECs optimal growth and formation of a monolayer were observed when the ECs were cultured on fibronectin or gelatin substrates in the presence of EC growth factor and heparin. Under these conditions confluent cultures of macrovascular ECs reached maximal cell densities of 1,400–1,900 ECs/mm², whereas microvascular ECs reached maximal cell densities of about 700–900 ECs/mm². The cell cycle times calculated from the population-doubling time and the stathmokinetic index, respectively, amounted to 63 and 83 h for microvascular ECs, 33 and 35 h for venous macrovascular ECs, and 29 and 35 h for arterial macrovascular ECs.

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