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 > 12,000) to survive, a small molecular weight (< 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 non-dialyzable 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.     

Opposing effects of dexamethasone on the clonal growth of granulocyte and macrophage progenitor cells and on the phagocytic capability of mononuclear phagocytes at different stages of differentiation

Dexamethasone, a synthetic glucocorticosteroid, was shown to modulate the colony-stimulating factor-dependent clonal growth of myeloid progenitor cells in semisolid agar cultures, enhancing the formation of granulocyte colonies (50–100%) and suppressing the formation of macrophage colonies (75–97%). Modulation of the pattern of myeloid colony formation by dexamethasone (12–125 nM) was brought about when the steroid was administered to 6-day cultures at the time of culture initiation and up to 72 hr later. Dexamethasone inhibited myeloid cell proliferation when administered to 5-day liquid cultures at culture initiation and up to 96 hr later. Dexamethasone (12–250 nM) also enhanced the phagocytic activity of bone marrow-derived mononuclear phagocytes toward heat-killed (HK) yeast cells (up to 100%) and IgG-coated sheep red blood cells (up to 60%). Enhancement of the phagocytic capability depended critically on the stage in culture at which dexamethasone was administered. Exposure to dexamethasone for 28 hr up to 96 hr of 96-hr cultures of bone marrow cells did not lead to a modulation of phagocytic activity of the developing mononuclear phagocytes. The presence of dexamethasone during the critical period of 96 hr to 120 hr after culture initiation led to an enhanced phagocytic capability, which was statistically significant already 12 hr after the administration of the glucocorticoid. Dexamethasone induced an enhanced phagocytic activity when administered at any time after culture initiation provided that it was in culture during this critical period. When added at 120 hr of culture, dexamethasone no longer enhanced the phagocytic capability of mononuclear phagocytes and when added later than 156 hr of culture suppressed it. Dexamethasone also suppressed (up to 68%) the phagocytic capability of resident and elicited peritoneal macrophages. The results suggest that glucocorticoids shift the balance of granulocyte vs. macrophage formation at early stages of precursor cell differentiation. Reduction in mononuclear phagocyte growth and enhancement of its phagocytic capability might reflect accelerated differentiation/maturation steps. The inhibitory effect of dexamethasone on macrophage formation and on the phagocytic capability of mature mononuclear phagocytes and peritoneal macrophages might be a relevant aspect of the in vivo immune suppression encountered after glucocorticoid administration.     

Expression of gamma-interferon receptor in murine bone marrow-derived macrophages associated with macrophage differentiation: evidence of gamma-interferon receptors in the regulation of macrophage proliferation

The effect of purified, recombinant murine gamma interferon (IFN-gamma) on the regulation of macrophage proliferation induced by colony-stimulating factor 1 (CSF-1) was investigated. Although both hemopoietic stem cells (GM-CFC) and tissue-derived peritoneal exudate macrophages (PEM) proliferated in response to CSF-1, the more mature PEM were much more sensitive to an antiproliferative effect of IFN-gamma. The role of IFN-gamma receptor expression and its relationship to growth inhibition was examined. Bone marrow cells as a whole did not exhibit an appreciable amount of IFN-gamma receptor binding activity. Likewise, nonadherent (NA) cells derived from CSF-1-stimulated bone marrow cultures displayed low levels of IFN-gamma receptor binding activity. On the contrary, more mature adherent (AD) cells (monocytes/macrophages) from the same culture exhibited high levels of IFN-gamma receptor binding activity, which continued to increase with culture time. The elevated IFN-gamma binding activity is due to an increase in total receptor number rather than the binding affinity as judged by Scatchard analysis. Similar to the relationship between PEM and GM-CFC, more mature AD cells were also more susceptible to the inhibitory effect of IFN-gamma on CSF-1-induced proliferation than their less mature NA counterparts. The fact that the sensitivity to IFN-gamma correlated well with the expression of existing IFN-gamma receptors strongly suggests that the inhibitory effect is mediated through IFN-gamma receptors. This study shows that the expression of IFN-gamma receptors in mononuclear phagocytes may not only represent one of the phenotypic parameters acquired by the growing macrophages during the process of differentiation, but may play some role in controlling proliferation.     

Peritoneal exudate cells. I. Growth requirement of cells capable of forming colonies in soft agar

Mouse peritoneal exudate cells induced by thioglycollate medium can form colonies in soft agar with a plating efficiency of about 5% (0.6%–10%). Cells from an unstimulated peritoneal cavity form no colonies or have a plating efficiency of less than 0.001 %. These colony-forming cells from the peritoneal exudate are similar to bone marrow colony-forming cells in vitro in that they both require a substance(s) present in conditioned medium from L-cells or mouse embryo fibroblasts or the serum from endotoxin-treated mice for the initiation and the continuation of their growth. However, peritoneal exudate colony-forming cells have a much longer initial lag period (10–14 days) and can survive longer in the absence of L-cell conditioned medium than bone marrow colony-forming cells. Only mononuclear cells, presumably macrophages, are observed in peritoneal exudate colonies, whereas bone marrow cell colonies contain both polymorphonuclear cells and macrophages.     

Effect of mononuclear phagocytes on the differentiation of syngeneic, allogeneic and xenogeneic hematopoietic stem cells

The colony formation in spleen of lethally irradiated syngeneic or hybrid recipients was studied after transplantation of bone marrow cells, with or without macrophages from lymph nodules or from peritoneal cavity of mice, cells of macrophage-like cell line J-774, and monocytes from peripheral blood of healthy donors. The direction of stem cell differentiations in the presence of all the types of mononuclear phagocytes was seen to change from mainly erythroid to mainly myeloid one. The ratio of erythroid to myeloid colonies became equal to 0.5-0.9 instead of 2.0, when bone marrow cells were injected with equivalent quantity of mononuclear phagocytes. This new regulatory function of mononuclear phagocytes is discussed.     

Development and characterization of monoclonal antibodies to murine macrophage colony-stimulating factor

The macrophage-specific CSF (CSF-1), purified from murine L cell-conditioned medium, supports the in vitro proliferation and survival of various murine mononuclear phagocyte colony-forming cells. In this report we describe the production and functional characterization of two monoclonal antibodies (mAb) to CSF-1 obtained from rat X rat hybridomas. These two mAb are functionally distinct and recognize different epitopes on CSF-1. The mAb 5A1 binds to and inhibits the biologic function of CSF-1, and the second mAb (D24) binds CSF-1 but does not neutralize its biologic activity. The mAb 5A1 inhibits colony formation of tissue mononuclear phagocyte colony-forming cells as well as the committed bone marrow stem cells for both granulocytes and monocytes. The extent of colony inhibition by mAb 5A1 is dependent on the tissue origin of colony-forming cells. CSF-1 complexed with mAb 5A1 does not bind to its cell surface receptor of peritoneal exudate macrophages, and mAb 5A1 does not complex with cell-bound CSF-1. Although both bone marrow cell-derived macrophages and J774.1 macrophages bind CSF-1, mAb 5A1 inhibits the proliferation of only bone marrow cell-derived macrophages. The non-neutralizing mAb D24 does not block binding of CSF-1 to its cellular receptor, and it recognizes cell-bound CSF-1.     

Infection of macrophages with Friend virus: relationship to the spontaneous regression of viral erythroleukemia.

Resident peritoneal macrophages from normal mice were refractory to infection with the RFV or conventional strains of Friend virus (FV). Stimulation of DNA synthesis in the macrophage population by induction of an exudate in vivo or treatment in vitro with macrophage colony-stimulating factor resulted in productive infection following exposure to virus. Similarly, normal resident macrophages did not become infected in vivo following transfer to leukemic mice, while exudate macrophages did become infected. Bone marrow macrophage stem cells were stimulated to replicate and mature in clonal agar cultures in the presence of colony-stimulating factor. These replicating stem cells could be infected with RFV, as shown by virus production in the resultant progeny macrophages. Transfer of normal resident peritoneal macrophages to leukemic progressor mice caused regression of the disease. In contrast, transfer of normal bone marrow cells was ineffective in causing leukemia regression. During erythroleukemogenesis induced by RFV, macrophage precursor cells in all of the mice became infected with virus. In mice with a progressive and lethal leukemia, mature end-stage macrophages were produced which were also infected with virus. In mice in which the leukemia would later spontaneously regress, the infected stem cells were eliminated and the marrow became repopulated with uninfected cells. The resultant progeny macrophages which appeared in the peritoneal cavity were uninfected and thus capable of participating in or causing leukemia regression.     

Regulation of lymphocyte motility by macrophages: characterization of a lymphocyte migration inhibitory factor derived from a macrophage-like cell line

An inhibitory factor on lymphocyte migration was detected using a capillary random migration assay in the culture supernatant of peritoneal exudate macrophages cultured at concentrations greater than 8 x 10(6) cells/ml. After examining different macrophage-like cell lines, J774A.1 cells were found to produce this inhibitory factor, which was termed lymphocyte migration inhibitory factor (LMIF). The inhibitory effect of LMIF on the migration of spleen lymphocytes, thymocytes, and bone marrow cells was determined. The migration of thymocytes was more sensitive to LMIF than was the migration of spleen lymphocytes and bone marrow cells. Interestingly, when the effect of LMIF was tested on the migration of spleen T cells and B cells, T cells were more sensitive than B cells. When the thymocytes were separated by peanut agglutinin into mature and immature thymocytes, the migration of mature thymocytes was more sensitive than that of immature thymocytes, the migration of mature thymocytes was more sensitive than that of immature thymocytes to the effect of LMIF, suggesting that the greatest effect of LMIF was on the migration of mature T cells. Partial purification of LMIF by ion-exchange and gel-filtration chromatography revealed that it is approximately 14,000 in molecular weight and could exist in either monomeric or dimeric forms. The possible role of this factor in an immune response is discussed.     

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.     

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

The binding and degradation of soluble guinea pig IgG₂ 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.     

Plasminogen activator-specific inhibitors in mouse macrophages: in vivo and in vitro modulation of their synthesis and secretion

Mouse resident peritoneal macrophages synthesize two plasminogen activator-specific inhibitors (PAI) that are functionally and antigenically related, but differ in their apparent Mr and oligosaccharide content. Most of the Mr 40,000 inhibitor can be recovered from the cell lysate, whereas the Mr 55,000 glycosylated PAI is preferentially secreted. The murine macrophage PAI are functionally similar and immunologically related to PAI synthesized and secreted by human monocytes-macrophages, and to a PAI from human placenta (PAI-2). PAI production by murine mononuclear phagocytes can be modulated both in vivo and in vitro. Bone marrow-derived macrophages do not produce detectable PAI, whereas inflammatory macrophages obtained from thioglycollate-induced peritoneal exudates produce only low levels of PAI. In cultures of resident peritoneal macrophages, phorbol myristate acetate and cholera toxin increase the synthesis of the Mr 55,000 secreted PAI, whereas dexamethasone decreases the synthesis of both PAI; the production of PAI is also enhanced in the presence of macrophage colony-stimulating factor (CSF-1). The overall proteolytic activity of mononuclear phagocytes thus depends in part on the controlled synthesis and secretion of PAI. The balance between the production of plasminogen activators and of their inhibitors could be critical in determining the level of plasminogen-dependent extracellular proteolysis associated with different phases of the inflammatory response.     

Microglial mitogens are produced in the developing and injured mammalian brain

The central nervous system produces growth factors that stimulate proliferation of ameboid microglia during embryogenesis and after traumatic injury. Two microglial mitogens (MMs) are recovered from the brain of newborn rat. MM1 has an approximate molecular mass of 50 kD and a pI of approximately 6.8; MM2 has a molecular mass of 22 kD and a pI of approximately 5.2. These trypsin-sensitive proteins show specificity of action upon glia in vitro serving as growth factors for ameboid microglia but not astroglia or oligodendroglia. Although the MMs did not stimulate proliferation of blood monocytes or resident peritoneal macrophage, MM1 shows granulocyte macrophage colony-stimulating activity when tested upon bone marrow progenitor cells. Microglial mitogens may help to control brain mononuclear phagocytes in vivo. The MMs first appear in the cerebral cortex of rat during early development with peak levels around embryonic day E-20, a period of microglial proliferation. Microglial mitogens are also produced by traumatized brain of adult rats within 2 d after injury. When infused into the cerebral cortex, MM1 and MM2 elicit large numbers of mononuclear phagocytes at the site of injection. In vitro study shows that astroglia from newborn brain secrete MM2. These observations point to the existence of a regulatory system whereby secretion of proteins from brain glia helps to control neighboring inflammatory responses.     

Effect of doxorubicin on the functional state of the mononuclear phagocyte system]

The response of the system of mononuclear phagocytes (SMP) to doxorubicin, an antitumor antibiotic, most widely used in oncological care, was studied. It was shown that a single intraperitoneal administration of doxorubicin to CBA mice in the maximum tolerance doses induced suppression of absorptive SMP capacity and increased IL-I secretion by the bone marrow and peritoneal macrophages both in the stimulated and spontaneous tests in early periods after cytostatic administration. There was a significant rise in the ability of SMP bone marrow elements to respond to the macrophage activating factor, as well as an increase in the cytotoxic activity of bone marrow and peritoneal macrophages.     

Cytochemical localization of glucose-6-phosphatase in rabbit mononuclear phagocytes during differentiation

Cytochemical and biochemical investigations have revealed glucose-6-phosphatase (G-6-Pase) activity in Kupffer cells of the liver. To determine whether other mononuclear phagocytes are also reactive for G-6-Pase, rabbit bone marrow, blood, and alveolar macrophages were tested for G-6-Pase by a modified Wachstein-Meisel method and prepared for electron microscopy. Some mononuclear phagocytes from all three tissues were intensely reactive; others were unreactive. In promonocytes, monocytes, and alveolar macrophages, reaction product for the enzyme was localized throughout all cisternae of the endoplasmic reticulum (ER) and the perinuclear cisternae, but it was absent from the Golgi complex, lysosomes, and occasional smooth tubular channels. These results indicate that mononuclear phagocytes at all stages of development contain cytochemically demonstrable G-6-Pase and that the distribution of the enzyme is not altered during their differentiation from immature cells in the bone marrow to mature macrophages in the lung.     

Selective induction of enhanced complement receptor 3 activity on murine macrophages

A high proportion of murine resident peritoneal macrophages bear complement receptors 1 and 3 (CR1, CR3) which bind C3b and iC3b components of complement, respectively. By contrast, macrophages derived from bone marrow, blood, and the elicited peritoneal exudate are predominantly CR1+3. To determine if the microenvironment of the normal peritoneal cavity influences CR3 phenotype, we studied the effects of lavage from the cavity on cultures of primary peritoneal exudate macrophages, and on macrophages derived from progenitors in the bone marrow, blood, and peritoneal exudate. The cell-free peritoneal lavage (CFPL), after 24 hr of culture, induced CR3 on primary and culture-derived populations of peritoneal exudate macrophages but had no effect on the CR3 phenotype of macrophages derived from bone marrow or blood. The CR3-inducing activity in CFPL was abolished by heating at 70 degrees C for 30 min and by trypsin, and was not affected by adsorption with EA(IgM)iC3b indicator cells, demonstrating that it is not soluble CR3. Finally, exudate macrophages exposed to CFPL required at least 24 hr before they expressed CR3; such macrophages regenerated CR3 after the receptors were removed by trypsin. The selective effect of the activity in CFPL for peritoneal exudate macrophages indicates that the local microenvironment of the peritoneal cavity can influence the expression of CR3.     

Interferon-gamma inhibits proliferation, but not commitment, of murine granulocyte-macrophage progenitors.

We investigated the effects of interferon gamma (IFN-gamma) on the growth of murine hematopoietic progenitors. IFN-gamma inhibited granulocyte colony-stimulating factor (G-CSF)- and interleukin-3 (IL-3)-dependent colony growth by granulocyte-macrophage (GM) progenitors derived from the bone marrow cells of normal mice. However, the number of IL-3-dependent GM colonies formed by the bone marrow cells of 5-fluorouracil (5-FU)-treated mice was not influenced by the addition of IFN-gamma. Replating experiments suggested that IFN-gamma suppressed GM colony growth directly and that it exerted an inhibitory effect on the proliferation, but not on the commitment, of GM progenitors. In contrast, IFN-gamma failed to suppress colony growth by mast cell progenitors. Erythroid and megakaryocytic progenitors exhibited different responses to IFN-gamma depending on mouse strains. These results suggest that potent negative regulators are not always inhibitors of hematopoietic progenitors.     

Onset of apoprotein E secretion during differentiation of mouse bone marrow-derived mononuclear phagocytes

A number of macrophage functions were sequentially expressed when the bone marrow precursors of mononuclear phagocytes differentiated in culture in the presence of a specific growth factor, colony-stimulating factor-1. We have defined the expression of apoprotein E (ApoE), a major secreted protein of resident peritoneal macrophages, during maturation of adherent bone marrow-derived mononuclear phagocytes into macrophages. By 5 d the bone marrow macrophages were active secretory cells, but few cells contained intracellular immunoreactive ApoE, and little, if any, ApoE was secreted. ApoE secretion was initiated at 9 d, and this correlated with an increase in the percentage of macrophages containing intracellular ApoE. The onset of ApoE secretion was selective, and little change occurred in the other major secreted proteins detected by [35S]methionine incorporation. In parallel, the high rate of plasminogen activator secretion, which peaked at 7 d, decreased markedly. ApoE secretion was not associated with altered expression of the macrophage surface antigen, Ia, or with secretion of fibronectin. Virtually all cells in independent colonies of bone marrow- derived macrophages eventually expressed ApoE. The proliferating monocyte/macrophage-like cell lines P388D1, J774.2, WEHI-3, RAW 264.1, and MGI.D+ secreted little or no ApoE. These data establish that ApoE secretion is developmentally regulated.     

Distinct bone marrow precursors for mononuclear phagocytes expressing high and low 5' -nucleotidase activity

Using a cytochemical assay we were able to show that the peritoneal macrophage population of normal nontreated mice (resident peritoneal macrophages) exhibits a heterogeneity with regard to the expression of the activity of the ecto-enzyme 5′-nucleotidase (5′-N). About 75% of the macrophages express high enzymic activity whereas the remaining 25% express low 5′-N activity. Macrophages accumulating in the peritoneum as a result of an inflammatory response are predominantly of the low activity type. In vitro activation of resident peritoneal macrophages by lymphokines does not result in a decrease in the number of macrophages expressing high enzymic activity though the level of the enzymic activity of these cells is reduced by about 36%. Bone marrow derived mononuclear phagocyte colonies developing in vitro, under liquid culture conditions, from bone marrows of normal mice can be divided into three types with respect to their expression of 5′-N activity: (1) high activity colonies–relatively small colonies in which all the cells express high 5′-N activity (about 20% of the colonies); (2) low activity colonies – relatively large colonies in which all the cells express low 5′-N activity (about 70% of the colonies); and (3) mixed colonies–relatively large colonies in which all the cells express low enzymic activity except for about 8% of cells located at the periphery of the colonies which express high enzymic activity (about 10% of the colonies). During an inflammatory response the frequency of the high activity colonies is significantly reduced. Our results provide evidence for distinct bone marrow precursors for mononuclear phagocytes expressing high and low 5′-N activity and suggest that (1) the resident macrophages derive from a subpopulation of bone marrow precursor cells developing in vitro into high 5′-N activity mononuclear phagocytes, and (2) during an inflammatory response there is a preferential expansion of clones of the low enzymic activity phenotype.     

Control of lipoprotein lipase secretion in mouse macrophages

The regulation of secretion of lipoprotein lipase (LPL) was studied in in vitro-derived mouse bone marrow macrophages (BMM), peritoneal exudate and resident macrophages and in the macrophage-like tumor cell line J774.1. BMM in cultures initiated with low concentrations of bone marrow cells (LC-BMC cultures) secrete more LPL per cell than BMM in cultures initiated with high concentrations of bone marrow cells (HC-BMC cultures). The suppressed state of LPL secretion in HC-BMC cultures could be alleviated by the addition of a colony-stimulating factor source (L-cell-conditioned medium; L-CM) onto the culture medium or exchanging the medium of HC-BMC cultures with medium from LC-BMC cultures for short periods (4 h). Addition of L-CM increased LPL secretion also in LC-BMC cultures. Addition of L-CM to fresh culture medium had little or no effect, suggesting that, in addition to requirement for L-CM, optimal expression depended also on factors released by the growing cells, probably providing optimal growth conditions. L-CM enhanced LPL secretion by thioglycollate-elicited peritoneal macrophages and had no effect on LPL secretion by resident peritoneal macrophages. Secretion of LPL from adherent J774.1 cells showed a biphasic effect. Secretion increased with cell density up to the point when growth inhibition was observed. In dense cultures in which cell proliferation was almost arrested, LPL secretion was remarkably suppressed (80-90%). Change of medium of dense cultures to fresh medium or medium conditioned by sparse cultures (for the last 4 h of culture) led to enhancement of LPL secretion to levels similar to those optimally expressed by sparse cultures. L-CM did not enhance LPL secretion from J774.1 cells. Dense cultures of both BMM and J774.1 cells did not contain a stable inhibitor of LPL secretion and medium from sparse cultures did not contain an inducer of LPL secretion. The data suggest that proliferating macrophages secrete large amounts of LPL, whereas in nonproliferating, quiescent cells, this activity is much reduced. L-CM enhances LPL secretion in quiescent BMM and peritoneal exudate cells to levels expressed by proliferating cells. Since this effect is already expressed after a 4 h incubation period, it is not dependent on cell cycling but could be one of the early responses to this macrophage mitogen. In J774.1 cells, a change of medium is a sufficient signal for enhancement of LPL secretion in quiescent cells.     

Tumor-promoting phorbol esters inhibit the binding of colony-stimulating factor (CSF-1) to murine peritoneal exudate macrophages

L-cell colony-stimulating factor (CSF-1) is a sialoglycoprotein of molecular weight 70,000 daltons that specifically stimulates macrophage colony formation by single committed cells from normal mouse bone marrow and by various classes of more differentiated tissue-derived mononuclear phagocyte colony-forming cells (Stanley et al., 1978). CSF-1 interacts with target cells by direct and specific binding to membrane receptors (CSF-1 receptors) that are present only on cells of the mononuclear phagocyte series and their precursors. We studied the effect of tumor-promoting phorbol esters on the binding of 125I-labeled CSF-1 (125I-CSF-1) to murine peritoneal exudate macrophages (PEM). Biologically active TPA (12-O-tetradecanoyl phorbol-13-acetate) inhibits the binding of 125I-CSF-1 to its receptor on PEM. This inhibition exhibits temperature, time, and concentration dependence. At 37 degrees C, maximum inhibition occurred at about 10(-7) M; inhibition was 50% at 5 X 10(-9) M. At 0 degrees C, the inhibitory activity of TPA is diminished. The action of TPA on PEM is transient. Treated cells recover their 125I-CSF-1-binding activity whether TPA is later removed or not. The process of recovering CSF-1-binding activity is completely blocked by the addition of cycloheximide. When several phorbol derivatives were tested for their inhibitory activities, only biologically active phorbol esters were found to possess such activities. Furthermore, the inhibitory activities of various phorbol esters are proportional to their tumor-promoting activities. Inhibition appears to be due to a reduction in the total number of available CSF-1 receptors rather than a decrease in receptor affinity.