The present communication concerns a quantitative study on the production and kinetics of mononuclear phagocytes during an acute inflammatory response as compared with the steady-state condition. During an acute inflammation induced by an intraperitoneal injection of NBCS, the peritoneal macrophages increase 2.5 times and there is a concomitant threefold increase of the monocytes in the peripheral blood. This increase of the peritoneal macrophages could be caused by a local proliferation of these cells or by the recruitment of monocytes from the circulation. The results of the in vitro and pulse-labeling studies demonstrate that the mitotic activity of the peritoneal macrophages is not increased during the inflammatory response, which indicates that the increase in the number of these cells is not due to local proliferation. Evidence is also presented that the small proportion (maximally 4%) of peritoneal macrophages that synthesize DNA are very recently arrived from the circulation. In agreement with this is the finding that a small number (less than 3%) of the peripheral blood monocytes are capable of synthesizing DNA. Since proof was obtained that the macrophages in the inflammatory peritoneal exudate originate from peripheral blood monocytes and the number of these cells in the circulation was also augmented, an increased formation of monocytes in the bone marrow was expected. Because increased monocyte production could be brought about by an increased number of promonocytes and/or an acceleration of the mitotic activity of the promonocytes, the various parameters of the cell cycle of these cells were determined. In normal mice the DNA-synthesis time of the promonocytes was 11.8 h, the cell cycle time 16.2 h, and the G1 + G2 + M phases 4.4 h. During the first 12 h of the inflammatory response a significantly shorter DNA-synthesis time (7.6 h) and cell cycle time (10.8 h) was found. At 24 h, these values approximated those found in normal mice. Next, both the total production and the rate of production of the monocytes were calculated and compared for both conditions. This computation showed that the total production of labeled monocytes during the first 48 h of an acute inflammation was 64% greater than in normal mice. The rate of production, calculated in two ways (i.e., from the data of the total production and also from the data of the cell cycle time together with the total number of promonocytes) complemented each other very well. During the first 12 h of the inflammatory response the production rate was increased 1.5 times and then leveled off, reaching almost the normal rate after 24 h. Furthermore, the excellent agreement between the results of the two methods of calculation for the normal steady state confirmed once more that the promonocyte is the direct precursor cell of the monocyte, giving rise to the two monocytes after each division. The kinetics of the monocytes in the peripheral blood was also altered during the inflammatory response. During the first 48 h, twice the normal number of labeled monocytes went from the bone marrow to the peripheral blood and twice the normal number also left the circulation. Furthermore, at least 70% of this increased number of labeled monocytes leaving the circulation migrated into the inflammatory exudate of the peritoneal cavity, leading to a roughly 11-fold increase of labeled peritoneal macrophages.

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Isolation of macrophages from the peritoneal cavity, liver, and lung of normal mice has enabled us to characterize these cells with respect to their morphology, cytochemical characteristics, and functional properties (1-3). Furthermore, their origin, proliferative behavior, and turnover have been investigated (1-6). Chimera studies have shown that macrophages derive from a precursor cell in the bone marrow (7), and kinetic studies with [SH]thymidine as cell marker have demonstrated that the immediate ancestor of the macrophage is the circulating monocyte. These kinetic studies have also provided quantitative information about the influx of monocytes into a body cavity (e.g., the peritoneal cavity) (4) or an organ (e.g., liver, lung) (5, 6).Morphological studies have shown that the spleen contains a large number of macrophages (8, 9), but the origin and kinetics of these cells have not been studied in detail. It is highly probable that spleen macrophages too derive from circulating monocytes, but local formation of macrophages by dividing precursor cells is possible in rodents, where the spleen continues to be a hematopoietic organ in the adult animal.The present report concerns a study on the cytochemical and functional characteristics of spleen macrophages and the origin and kinetics of these cells, using macrophage suspensions obtained by mechanical treatment and enzyme digestion. Materials and MethodsAnimals. Specific pathogen-free male Swiss mice (The Central Institute for the Breeding of Laboratory Animals, TNO, Zeist, The Netherlands) weighing 25-30 g were used.Isolation of Spleen Macrophages. The animals were anesthetized with 5 mg phenobarbital (Abbott NV, Amsterdam) injected intraperitoneaily. The abdomen was opened, the diaphragm exposed and pierced, and, after collapse of the lungs, a piece of the thoracic cage was excised. The heart was punctured with a 22-gauge needle on a 10-ml syringe containing 0.6 mM EDTA in phosphate-buffered saline. ~4 ml was injected slowly while the abdominal aorta was clamped; next, the aorta and vena cava in the abdomen were cut and the remaining EDTA-saline solution was injected slowly; during this perfusion the spleen became slightly paler. The organ was then removed, weighed, washed once in medium 199 (M.A. Bioproducts, Waikersviile, MD), and cut into small pieces (~1 ramS). The fragments were incubated in 10 ml 0.1% collagenase (126 U/rag; Worthington Biochemical Corp., Freehold, NJ) in Hanks' balanced salt solution (pH 7.4) for 1 h in a 37°C water bath under constant stirring. During incubation, the pH was monitored and if necessary adjusted with 0.1 N sodium hydroxide. The suspension was then drawn into and expelled from a syringe with a 19-gauge needle three times, filtered through two layers of gauze, and then centrifuged for 7 rain at 400 g. The pellet was washed twice J. ExP. MEo.

The mononuclear phagocytes of the bone marrow can be classified into two cell types, promonocytes and monocytes. The present study was performed to establish whether the promonocytes are the progenitors of the monocytes and to determine the kinetic characteristics of the promonocytes and monocytes in the bone marrow compartment. Both in vitro labeling studies with thymidine-3H and determination of the relative amount of DNA in the nuclei of individual cells showed that under normal steady-state conditions the promonocytes are proliferating cells and the monocytes, nondividing cells. In vivo labeling studies provided further evidence that the promonocytes are the progenitor cells of the monocytes. During the first 24 hr after labeling, the promonocytes showed a constant high level of labeling (about 70%). The mean grain count of these cells decreased with time. The labeling index of the monocytes of the bone marrow increased during the first 24 hr after in vivo labeling, but during the same period the mean grain counts remained almost constant, with values amounting to about half those of the promonocytes during the first 6 hr of the experiment. The data concerning the labeling indices and the percentage distribution ratio of the promonocytes and monocytes in the bone marrow, and the labeling indices of the peripheral blood monocytes are used to construct a model population. The results lead to the conclusions that the promonocytes are multiplicative cells and that both daughter cells arising from the division of a promonocyte are monocytes. The DNA-synthesis time found for the promonocytes is 13.6 hr. From this value, the average generation time was computed to be 19.5 hr.

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