Bone Marrow Mononuclear Phagocytes Research Articles

Porcine mononuclear phagocyte subpopulations in the lung, blood and bone marrow: dynamics during inflammation induced byActinobacillus pleuropneumoniae

Mononuclear phagocytes (MP) are cells of nonspecific immunity, playing an essential role in defense against bacterial pathogens. Although various MP subpopulations have been described in the pig, relations among these populations in vivo are unknown to date. The present study was aimed at describing porcine MP subpopulations infiltrating inflamed tissue of pigs under in vivo conditions. Actinobacillus pleuropneumoniae (APP) infection was used to induce an inflammatory response. CD172α, CD14, CD163, MHCII and CD203α cell surface molecules were used to identify MP by flow cytometry. Changes in MP subpopulations in the peripheral blood (PB) and bone marrow (BM) compartments along with the analysis of MP appearing in the inflamed lungs were assessed to elucidate the possible origin and maturation stages of the infiltrating MP. The MP population migrating to the inflamed lungs was phenotype CD14+ CD163+ CD203α+/− MHCII+/−. Concomitantly, after APP infection there was an increase in the PB MP CD14+ CD163+ CD203α− MHC II− population, suggesting that these cells give rise to inflammatory monocytes/macrophages. The CD203α and MHCII molecules appear on these cells after leaving the PB. In healthy animals, the BM MP precursors were represented by CD14− CD163− cells maturing directly into CD14+ CD163− that were then released into the PB. After infection, an altered maturation pathway of MP precursors appeared, represented by CD14− CD163− CD203α− MHCII− MP directly switching into CD14+ CD163+ CD203α− MHCII− MP. In conclusion, two different MP maturation pathways were suggested in pigs. The use of these pathways differs under inflammatory and noninflammatory conditions.

Enrichment of human bone marrow mononuclear phagocytes and characterization of macrophage subpopulations by immunoenzymatic double staining.

In order to isolate and enrich bone marrow mononuclear phagocytes, we performed magnetic-activated cell sorting using beads coupled to a monoclonal antibody directed against the monocyte/macrophage surface molecule CD14. Colocalization of antigens in single cells was achieved by combining an alkaline phosphatase-anti-alkaline phosphatase and an avidin-biotin complex immunoassay, avoiding the use of peroxidase. Bone marrow macrophages were first labelled by the monoclonal antibody PG-M1 (anti-CD68). Subsequently, cytoplasmic and/or surface double staining by the monoclonal antibodies against HLA-DR and Mac-2 antigen or the lectin GSA-I-B4 was carried out. Whereas HLA-DR was co-expressed by the great majority of PG-M1+ macrophages (84.9%+/-6.9%), only a subpopulation exhibited Mac-2 (69.9%+/-5.9%) antigen or galactoside structures detected by GSA-I-B4 (65.0%+/-6.7%). The latter result differed only slightly from the percentage of GSA-I-B4+ macrophages determined in a previous comparative immunomorphometrical study. Therefore, using our method of isolation and enrichment by magnetic-activated cell sorting, only a negligible portion of macrophages is apparently stimulated, as shown by GSA-I-B4 staining. This methodology seems to be a valuable tool for further studies on the monocyte-macrophage system.

Alterations in the morphology and functional activity of bone marrow phagocytes following benzene treatment of mice

Benzene is a well-established hematotoxin that affects developing leukocytes and erythrocytes as well as bone marrow stromal cells. In the present studies we analyzed the effects of benzene on the morphology and functional activity of bone marrow phagocytes. Male Balb c mice were treated with benzene (660 mg/kg) once per day for 3 days. Bone marrow cells were then isolated and fractionated by density gradient centrifugation. Using highly sensitive techniques in flow cytometry/cell sorting, we found that we could separate three distinct populations of bone marrow cells that differed with respect to size and density. Monoclonal antibody binding and cell sorting revealed a large, dense population that consisted predominantly of granulocytes, a smaller, less dense population of lymphocytes, and a population of intermediate size and density consisting of mononuclear phagocytes and precursor cells. Differential staining of sorted mononuclear phagocytes revealed that benzene treatment of mice caused a marked increase in the number of mature, morphologically activated macrophages in the bone marrow. Benzene treatment of mice also resulted in enhanced chemotaxis and production of hydrogen peroxide by bone marrow granulocytes and mononuclear phagocytes. In contrast, treatment of mice with the combination of hydroquinone and phenol (50 mg/kg each, 1×/day, 3 days), two metabolites of benzene, resulted in a significant ( p ≤ 0.02) depression of granulocyte chemotaxis and had no effect on hydrogen peroxide production by bone marrow phagocytes compared to cells from control animals. Taken together these results demonstrate that benzene causes increased differentiation and/or activation of phagocytes in the bone marrow.

Effect of Bone Marrow Mononuclear Phagocytes on the Bone Matrix-induced Bone Formation in Rats

Experimental ulnar bone defects in rats were grafted with freshly isolated whole bone marrow cells; bone marrow mononuclear phagocytes (macrophages); or both types of marrow cell preparations in combination with demineralized bone matrix gelatin (BMG). In the absence of BMG, the osteogenic performance of the marrow cell preparations was superior to that of the macrophages. In the presence of BMG (composite grafts), their osteogenic potential was nearly identical and significantly improved the level of bone formation stimulated by implants of BMG alone. The results encourage speculation and further research on sequential activities of bone marrow monocyte-macrophage (osteoclast) lineages and marrow stromal (osteoprogenitor) cell in bone morphogenetic protein (BMP)-induced regeneration.

The effect of glucocorticosteroids on bone marrow mononuclear phagocytes in culture

The effect of hydrocortisone and dexamethasone both in vivo and in vitro was studied in mouse bone marrow cultures in methylcellulose and in two liquid culture systems, one using Leighton tubes with a flying coverslip to grow adherent colonies and the other using Teflon culture bags to obtain suspension cultures. Although the total number of nucleated bone marrow cells was not greatly influenced by glucocorticosteroid treatment of the mice, a marked decrease in the number of colony-forming units and of mononuclear phagocytes was observed. Inhibition of colony growth in methylcellulose and of growth of mononuclear phagocytes in Teflon culture bags also occurred when glucocorticosteroids were added to in vitro cultures. Both drugs caused an almost complete inhibition of the growth of adherent colonies, 3Hthymidine labeling of the cells was correspondingly low. When the glucocorticosteroids were added to cultures pre-incubated for 5 days in the presence of conditioned medium, the 3Hthymidine labeling of macrophages and promonocytes was markedly reduced, whereas there was no change in the labeling of monoblasts.

Osteoclast formation from mononuclear phagocytes: role of bone-forming cells.

In a previous study, using co-cultures of embryonic bone rudiments stripped of periosteum, and mononuclear phagocytes of various sources, we found that multinucleated mineral-resorbing osteoclasts developed in vitro from radiosensitive mouse bone marrow mononuclear phagocytes (BMMP). (Burger, E. H., J. W. M. van der Meer, J. S. van de Gevel, C. W. Thesingh, and R. van Furth, 1982, J. Exp. Med. 156:1604-1614). In the present study, this co-culture technique was used to analyze the influence of bone-forming cells on osteoclast formation and bone resorption by BMMP or peritoneal exudate cells (PEC). BMMP or PEC were co-cultured with liver or dead bone, i.e., in the presence or absence of liver bone-forming cells. Mineral resorption and osteoclast formation were monitored via 45Ca release from prelabeled live or dead bone followed by histology. Osteoclasts developed from precultured BMMP as indicated by [3H]thymidine labeling, but only in live and not in dead bone. They formed readily from BMMP but only erratically, and after a longer culture period, from PEC. Macrophages from BMMP and PEC invaded live and dead bone rudiments but did not resorb the intact mineralized matrix. In contrast, ground bone powder was resorbed avidly by both cell populations, without formation of osteoclasts. We conclude that live bone-forming cells are required for osteoclast formation from progenitors. Live bone is only resorbed by osteoclasts, and not by macrophages. Osteoclast progenitors are abundant in cultures of BMMP but scarce in PEC, which makes a direct descendance of osteoclasts from mature macrophages unlikely.

In vitro formation of osteoclasts from long-term cultures of bone marrow mononuclear phagocytes.

The origin of osteoclasts was studied in an in vitro model using organ cultures of periosteum-free embryonic mouse long-bone primordia, which were co-cultured with various cell populations. The bone rudiments were freed of their periosteum-perichondrium by collagenase treatment in a stage before cartilage erosion and osteoclast formation, and co-cultured for 7 d with either embryonic liver or mononuclear phagocytes from various sources. Light and electron microscopic examination of the cultures showed that mineralized matrix-resorbing osteoclasts developed only in bones co-cultured with embryonic liver or with cultured bone marrow mononuclear phagocytes but not when co-cultured with blood monocytes or resident or exudate peritoneal macrophages. Osteoclasts developed from the weakly adherent, but not from the strongly adherent cells of bone marrow cultures, whereas 1,000 rad irradiation destroyed the capacity of such cultures to form osteoclasts. In bone cultures to which no other cells were added, osteoclasts were virtually absent. Bone-resorbing activity of in vitro formed osteoclasts was demonstrated by 45Ca release studies. These studies demonstrate that osteoclasts develop from cells present in cultures of proliferating mononuclear phagocytes and that, at least in our system, monocytes and macrophages are unable to form osteoclasts. The most likely candidates for osteoclast precursor cells seem to be monoblasts and promonocytes.

Binding and degradation of soluble immunoglobulin aggregates by mouse mononuclear phagocytes—Stimulation by colony-stimulating factor

The binding and degradation of soluble guinea pig IgG 2 aggregates by murine mononuclear phagocytes were studied. Bone marrow mononuclear phagocytes cultured in the presence of an embryonic fibroblast-conditioned medium (CM) degraded the aggregates to a much greater degree than did resident peritoneal macrophages. Binding and degradation by resident peritoneal macrophages were enhanced by culture in the presence of CM. Freshly harvested thioglycollate-induced peritoneal macrophages bound and degraded the aggregates to the same degree as the cultured bone marrow mononuclear phagocytes did. However, the thioglycollate-induced macrophages lost most of these capacities when cultured in vitro without CM. When CM was added to these cultures, the capacity to bind and degrade was restored in a dose-dependent fashion. To obtain the maximum effect, exposure to CM must be maintained for more than 2 days. The effect of CM could be reproduced with purified CSF-1. Taken together the results of this study indicate that Fc receptor expression is modulated by CSF-1.

Effects of serum fractions on the growth of mononuclear phagocytes.

The effects of serum fractions on the growth kinetics and colony formation of mononuclear phagocytes derived from mouse bone marrow, blood, and peritoneal cavity were investigated. Peritoneal exudate macrophages and blood monocytes required a factor(s) found to reside in the nondialyzable serum fraction (molecular weight greater than 12,000) to survive, a small molecular weight (less than 307) factor(s) with growth-stimulatory activity (GSA) contained in the dialyzable serum fraction, and the macrophage growth factor (MGF) for proliferation and colony formation. Fetuin, a major protein of fetal serum, was able to substitute the nondialyzable serum fraction. Macrophages cultured in medium containing MGF and the nondialyzable serum fraction for 6 days could be restored to full growth following the addition of the dialyzable serum fraction. In contrast, bone marrow mononuclear phagocytes cultured in the absence of the dialyzable serum fraction were capable of proliferating, though at a slower rate, and forming colonies. In addition, neither insulin nor hydrocortisone was capable of replacing the serum-dialyzable GSA nor able to enhance colony formation.

Macrophage growth inhibitors derived from the murine peritoneal cavity.

The murine peritoneal cavity contains factors that inhibit the in vitro growth and colony formation of macrophages. The inhibition of macrophage growth is not due to cell death. In the presence of inhibitors, the growth of colony-forming macrophages is suppressed, and small clusters are formed as a result of limited proliferation. The more mature mononuclear phagocytes (blood monocytes and peritoneal exudate macrophages) are more sensitive to the overall inhibitory effect of the peritoneal inhibitors than the less mature bone marrow mononuclear phagocytes. Furthermore, using dialysis and Amicon ultrafiltration, at least two inhibitors with differential inhibitory effects can be demonstrated. The colony formation of bone marrow mononuclear phagocytes is suppressed mainly by a protease-resistant, small molecular weight (less than 1,000) dialyzable inhibitor. In contrast, peritoneal exudate macrophages are sensitive to both the small molecular weight inhibitor and a protease-sensitive, large molecular weight (greater than 12,000), nondialyzable inhibitor. The data suggest a possible existence of a dual inhibitor control on the proliferation of mononuclear phagocytes in vivo. In addition, the in vitro cultured peritoneal exudate cells are capable of producing inhibitors that mimic the activity of the in vivo inhibitors.

The isolation and cultivation of rabbit bone marrow mononuclear phagocytes

Rabbit bone marrow cells were cultured in a liquid medium in the presence of high concentrations of serum but in the absence of other added exogenous stimulating substances. Conditions were established that allow the selective proliferation of the precursor cells of the mononuclear phagocytes and their differentiation into macrophages. These cells were identified both on the basis of their morphological appearance as well as on several other properties (presence of Fc receptors at their surface, rapid phagocytosis of latex or India ink, continuous secretion of lysozyme). Their proliferation is preceded by the rapid degeneration of all the other cell types, including the granulocytes that are no longer found after 4 days of culture. Macrophage precursors (monoblasts and promonocytes) proliferate and persist in suspension until the 8th–10th day of culture when they become adherent. Essentially pure populations of macrophages could be maintained in culture for at least 4 weeks.

Culture of mononuclear phagocytes on a teflon surface to prevent adherence.

A method is described for the culture of mononuclear phagocytes in suspension by incubation on a Teflon film to which the cells do not adhere. The characteristics of peritoneal macrophages, bone marrow mononuclear phagocytes, macrophage cell lines, and fibroblasts cultured in this way are similar to those observed after culture on glass or plastic surfaces. Culture of mononuclear phagocytes in Teflon film dishes has three important advantages: the cells can be easily harvested without damage, recovery is almost complete, and the cells are not functionally impaired. Thus, this method makes it possible to use cultured mononuclear phagocytes for many studied that could previously only be done in freshly collected cells.

Maturation and Interrelationship of Mouse Mononuclear Phagocytes in Bone Marrow, Peripheral Blood and Peritoneal Cavity in Terms of Erythrophagocytic Activity

Ingestion by mononuclear cells in bone marrow, peripheral blood and the peritoneal cavity of red cells treated with antibodies and various chemicals was studied in an attempt to characterize these cell lines during development and to clarify the interrelationship among them. Bone marrow mononuclear phagocytes became intensively phagocytic during the second day of culture, indicating that the maturation time from progenitor cells to macrophages was about 48 hr. Blood monocytes exhibited very active erythrophagocytic activity after 24 to 48 hr cell culture. Uptake of sensitized red cells by both the cell lines was markedly inhibited by IgG irrespective of time periods of cell culture. Majority of peritoneal lymphocyte-like cells (LLC) separated from other forms of mononuclear cells were shown to become capable of erythrophagocytosis with maturation time to macrophages between 24 to 48 hrs. Under non-stimulated steady state, uptake of sensitized red cells by peritoneal macrophages was far less sensitive to IgG inhibition than that by bone marrow mononuclear phagocytes and blood monocytes, suggesting that IgG receptor does not play main role for the erythrophagocytosis by peritoneal macrophages. Peritoneal cells collected under inflammatory stimulus exhibited a character similar to bone marrow mononuclear phagocytes and blood monocytes with regard to IgG receptor. It was suggested that under steady state peritoneal macrophages arise and mature from local proliferative pool with some different phagocytic mechanism from that of bone marrow mononuclear phagocytes.