Modulation of granulocyte colony-stimulating factor receptors on murine peritoneal exudate macrophages by tumor necrosis factor-alpha.

Modulation of granulocyte CSF (G-CSF) receptors on murine peritoneal exudate macrophages (PEM) by various cytokines was investigated. At 4 degrees C, 125I-G-CSF receptor binding on PEM reached a plateau after 6 h and was specifically competed by unlabeled human rG-CSF but not by other cytokines, including human rG-CSF-1, murine recombinant granulocyte-macrophage CSF, murine rIFN-gamma, human rIL-1 beta, and murine rTNF-alpha. 125I-G-CSF bound to PEM has a half-life of 30 min at 37 degrees C. Preincubation of PEM with murine rTNF, murine recombinant granulocyte-macrophage CSF, CSF-1, or G-CSF for 30 min at 37 degrees C resulted in partial reduction of 125I-G-CSF binding capacity, whereas IL-1 or IFN-gamma did not inhibit G-CSF binding. Further studies indicated that reduction of G-CSF binding caused by TNF was a dose- and time-dependent process and did not require FCS. The reduction was transient, and receptor binding was recovered by incubation at 37 degrees C for 8 h. The recovery of G-CSF binding was inhibited in the presence of cycloheximide. In addition, G-CSF binding studies suggested that the TNF-induced decrease in G-CSF binding to PEM was probably due to a reduction in receptor number rather than receptor affinity. Modulation of G-CSFR by TNF was also observed on nonelicited macrophages from various strains of mice. Our results demonstrate a physiologic response of G-CSFR on macrophages that is modulated by TNF. This phenomenon may play an important, as yet unknown, role in the macrophage inflammatory response.     

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.     

Lipopolysaccharide inhibits the binding of colony-stimulating factor (CSF-1) to murine peritoneal exudate macrophages

These studies demonstrate the potent effect of bacterial endotoxin (LPS) on the inhibition of iodinated colony-stimulating factor- (125I-CSF-1) binding by murine peritoneal exudate macrophages (PEM) from C3H/An and C57BL/6 mice. As small an amount as 0.1 ng/ml LPS is sufficient to cause a significant inhibitory effect; this effect is temperature-, time- and concentration-dependent. LPS, however, causes minimal or no inhibition of 125I-CSF-1-binding by PEM from LPS-resistant C3H/HeJ mice. Inhibition of 125I-CSF-1-binding does not appear to be a result of a direct occupancy by LPS of CSF-1 receptors present on the cell membrane and is most likely due to a progressive loss of available CSF-1-binding sites. The effect can be neutralized by the addition of the antibiotic polymyxin B, which binds to the lipid A portion of LPS. The action of LPS on PEM is transient; treated cells recover their 125I-CSF-1-binding activity whether or not LPS is later removed. The restoration of 125I-CSF-1-binding activity can be blocked completely by the addition of cyclohexamide. These findings suggest the rapid, LPS-induced disappearance of CSF-1 receptors from the cell surface may be related to the activation of macrophages by LPS.     

Binding, internalization and degradation of colony-stimulating factor by peritoneal exudate macrophages

Iodinated colony-stimulating factor produced by L-cells (125I-CSF-1) binds specifically to murine peritoneal exudate macrophages. At 37 degrees C, the cell-bound 125I-CSF-1 was internalized and degraded very rapidly, with the appearance of radioactive iodotyrosine in the medium. At 0 degree C, the cell-bound 125I-CSF-1 was not internalized and degraded, nor did it dissociate from the membrane. The internalization and degradation at 37 degrees C could be blocked or reduced by the presence of phenylglyoxal, methylamine and NH4Cl. The chemical nature of the CSF-1 binding site is polypeptide as judged by its sensitivity to trypsin treatment. After the binding and degradation of unlabeled CSF-1, the exudate cells were no longer able to rebind freshly added 125I-CSF-1, indicating the removal of CSF-1 binding site. The binding capacity of these cells, however, could be restored by prolonged incubation at 37 degrees C but not at 0 degrees C in culture medium containing fetal calf serum.     

Both granulocyte-macrophage CSF and macrophage CSF control the proliferation and survival of the same subset of alveolar macrophages

The effect of granulocyte-macrophage (GM)-CSF on the proliferation of murine pulmonary alveolar macrophages in vitro was investigated. About 20% of freshly isolated alveolar macrophages formed colonies in both liquid and soft agar cultures in the presence of GM-CSF. GM-CSF was also found to be capable of maintaining the survival of these colony-forming cells in vitro. Moreover, GM-CSF could substitute for CSF-1 in maintaining the survival of CSF-1-responding pulmonary alveolar macrophage colony-forming cells in the absence of CSF-1. The concentration of GM-CSF required for maintaining the survival of colony-forming cells without proliferation was much lower than that required for the proliferation of these cells in vitro. It also enhanced the CSF-1-dependent clonal growth of alveolar macrophages. These data suggest that the colony-forming cells that respond to GM-CSF are the same subset of macrophages that form colonies in the presence of CSF-1. GM-CSF did not inhibit the binding of 125I-CSF-1 to alveolar macrophages at 0 degrees C. However, the preincubation of macrophages with GM-CSF at 37 degrees C resulted in a transient down-regulation of CSF-1 binding activity.     

Erythropoietin causes down regulation of colony-stimulating factor (CSF- 1) receptors on peritoneal exudate macrophages of the mouse

We have shown that erythropoietin (epo), the primary regulator of erythrocyte formation, diminished the binding to peritoneal exudate macrophages (PEM) of the principal macrophage growth regulator, colony- stimulating factor (CSF-1). The effect of epo on 125I-CSF-1 binding was dose-dependent; at a concentration of 1-2 U of epo/ml (10(-10) M), CSF- 1 binding was almost completely suppressed. Erythropoietin did not compete with CSF-1 for occupancy of the latter's receptors. The effect of epo on CSF-1 binding occurred at 37 degrees C but not at 2 degrees C, and during the continuous exposure of PEM to epo at 37 degrees C we found that CSF-1 binding reached a nadir at 1 h and recovered to pre- exposure levels in 7 h. Our novel results are consistent with the notion that specific receptors for epo exist on the cell surface of PEM and that binding of epo sets in motion a series of cellular events resulting in the internalization of CSF-1 receptors. Thus epo causes down regulation of CSF-1 receptors on PEM. We have previously shown that epo causes suppression of CSF-induced granulocyte-macrophage colony formation by mouse bone marrow cells. The results we present here provide a possible mechanism for these results.     

Uptake and destruction of 125I-CSF-1 by peritoneal exudate macrophages

The binding and uptake of the colony-stimulating factor CSF-1 by peritoneal exudate macrophages (PEM) from lipopolysaccharide insensitive C3H/HeJ mice was examined at 2 degrees C, and at 37 degrees C. At 2 degrees C, 125I-CSF-1 was bound irreversibly to the cell surface. At 37 degrees C, 90% of the cell surface associated 125I-CSF-1 was rapidly internalized and subsequently degraded and the remaining 10% dissociated as intact 125I-CSF-1. Thus classical equilibrium or steady state methods could not be used to quantitatively analyze ligand-cell interactions at either temperature, and alternative approaches were developed. At 2 degrees C, the equilibrium constant (Kd less than or equal to 10(-13) M) was derived from estimates of the rate constants for the binding (kon congruent to 8 x 10(5) M-1 s-1) and dissociation (koff less than or equal to 2 x 10(-7) s-1) reactions. At 37 degrees C, the processes of dissociation and internalization of bound ligand were kinetically competitive, and the data was formally treated as a system of competing first order reactions, yielding first order rate constants for dissociation, koff = 0.7 min-1 (t1/2 = 10 min) and internalization, kin = 0.07 min-1 (t 1/2 = 1 min). Approximately 15 min after internalization, low-molecular weight 125I-labeled degradation products began to appear in the medium. Release of this degraded 125I-CSF-1 was kinetically first order over three half-lives (Kd = 4.3 x 10(-2) min-1, t1/2 = 16 min). Thus CSF-1 binds to a single class of receptors on PEM, is internalized with a single rate limiting step, and is rapidly destroyed without segregation into more slowly degrading intracellular compartments.     

Binding, internalization and degradation of colony-stimulating factor by peritoneal exudate macrophages

Iodinated colony-stimulating factor produced by L-cells (¹²⁵I-CSF-1) binds specifically to murine peritoneal exudate macrophages. At 37°C, the cell-bound ¹²⁵I-CSF-1 was internalized and degraded very rapidly, with the appearance of radioactive iodotyrosine in the medium. At 0°C, the cell-bound ¹²⁵I-CSF-1 was not internalized and degraded, nor did it dissociate from the membrane. The internalization and degradation at 37°C could be blocked or reduced by the presence of phenylglyoxal, methylamine and NH₄Cl. The chemical nature of the CSF-1 binding site is polypeptide as judged by its sensitivity to trypsin treatment. After the binding and degradation of unlabeled CSF-1, the exudate cells were no longer able to rebind freshly added ¹²⁵I-CSF-1, indicating the removal of CSF-1 binding site. The binding capacity of these cells, however, could be restored by prolonged incubation at 37°C but not at 0°C in culture medium containing fetal calf serum.     

Role of phorbol ester receptors in the 12-0-tetradecanoyl-phorbol-13-acetate (TPA)-induced down-regulation of colony-stimulating factor (CSF-1) binding to murine peritoneal exudate macrophages

Treatment of murine peritoneal exudate macrophages (PEM) by tumor-promoting phorbol esters (TPA) results in a rapid loss of binding activity to radioactive-labeled colony-stimulating factor ([125I]-CSF-1) on the cell surface. The inhibitory effect of TPA on PEM is transient; treated cells recover full [125I]-CSF-1 binding activity in less than 6 hr at 37 degrees C either in the presence or after the removal of added TPA. The role of phorbol ester receptors in the induction of [125I]-CSF-1 binding inhibition was studied. The biologically active ligand [3H]-phorbol 12,13-dibutyrate ([3H]-PDBu) bound specifically to cultured murine PEM. At 0 degree C, stable and equilibrium binding occurred after 2-3 hr. Scatchard analysis revealed linear plots with a dissociation constant and receptor number per cell of 20.9 nM and 3.9 X 10(5)/cell, respectively. Treatment of PEM with biologically active phorbol esters at 37 degrees C rapidly inhibited the binding activity of [3H]-PDBu on cell surface (down-regulation) and rendered these cells refractory to the TPA-induced [125I]-CSF-1 binding inhibition by the subsequent TPA treatment. The inhibition of phorbol ester binding activity on TPA-treated PEM is caused by a reduction in the total number of available phorbol ester receptors rather than by a decrease in receptor affinity as judged by Scatchard analysis. The disappearance of [3H]-PDBu binding activity is reversible and transient. However, unlike CSF-1 receptors the restoration of phorbol ester receptors on TPA-treated PEM is a very slow process; a prolonged incubation of up to 72 hr after the removal of TPA was required for PEM to regain fully its [3H]-PDBu binding activity. Furthermore, the degree of TPA-induced CSF-1-receptor down-regulation is closely associated with the number of available phorbol ester receptors present on PEM at the time of treatment. Thus, the refractoriness to TPA diminished as the phorbol ester receptors on PEM recovered. A 72-hr incubation time at 37 degrees C was needed for PEM to lose their refractoriness and again become fully sensitive to TPA-induced CSF-1-receptor down-regulation. This study provides evidence that the loss of CSF-1-receptors induced by TPA treatment requires the presence of phorbol ester receptors and proceeds presumably via a co-internalization of both CSF-1 and phorbol ester receptors; the refractoriness to TPA is thereby induced by a transient loss of available phorbol ester receptors.     

The interaction of 125I-colony-stimulating factor-1 with bone marrow-derived macrophages

The colony-stimulating factor, CSF-1, stimulates cultured quiescent murine bone marrow-derived macrophages (BMM) to enter DNA synthesis with a lag phase of 10-12 h. The binding, dissociation, internalization, and degradation of 125I-CSF-1 by BMM during the lag phase were investigated. Quiescent BMM express approximately 5 X 10(4) cell surface receptor sites/cell but contain additional cryptic sites (approximately 10(5)/cell) that can appear at the cell surface within 10 min at 37 degrees C. Studies of the binding reaction at both 2 degrees C (Kd less than or equal to 2 X 10(-13) M) and 37 degrees C (Kd approximately 4 X 10(-10) M) are consistent with the existence of a single class of cell surface sites. The disappearance of cell surface 125I-CSF-1 following a 2-37 degrees C temperature shift results from two, competitive, first order processes, internalization and dissociation. Internalization (t1/2 = 1.6 min) is 6 times more frequent than dissociation (t1/2 = 9.6 min). Following internalization, 10-15% of the intracellular CSF-1 is rapidly degraded whereas the remaining 85-90% is slowly degraded by a chloroquin-sensitive first order process (t1/2 greater than 3.5 h). These findings were confirmed and extended by studies of the uptake of 125I-CSF-1 at 37 degrees C. Following addition of 125I-CSF-1, cell surface receptors are rapidly down-regulated (t1/2 approximately 7 min) and their replacement does not commence until 20-60% of pre-existing surface receptor sites have disappeared. Despite receptor replacement, initially from the cryptic pool and later by de novo synthesis and/or receptor recycling (4 molecules/cell/s at steady state), the number of receptors at the cell surface remains low. The process results in the intracellular accumulation of large amounts of 125I-CSF-1 (greater than 10(5) molecules/cell) by BMM. Thus, whereas the kinetics of association, dissociation, and internalization of CSF-1 with BMM and peritoneal exudate macrophages are similar, BMM, which exhibit a higher proliferative response, degrade growth factor 12 times more slowly.     

Interferon-induced inhibition of receptor-mediated endocytosis of colony-stimulating factor (CSF-1) by murine peritoneal exudate macrophages

Apart from its characteristic antiviral activity, interferon (IFN) also exerts a variety of biologic effects on macrophages. We have studied the effect of IFN on the expression of the colony-stimulating factor receptors (CSF-1 receptors) by murine peritoneal exudate macrophages (PEM). At 37 degrees C, murine IFN decreased the expression of the CSF-1 receptor activity in a time- and dose-dependent fashion by PEM from both endotoxin-sensitive (C3H/Sn) and endotoxin-resistant strains (C3H/HeJ) of mice. Scatchard analysis from the binding data suggests that the decreased expression of CSF-1 receptors is a result of decreased number of receptors rather than a decreased binding affinity. When IFN was incubated with anti-IFN before the addition to cultures, the effect was completely abolished indicating that this activity resides in the same molecules as IFN. The suppressed CSF-1 receptor activity on PEM by IFN appeared to be stable. Removal of added IFN never resulted in a full recovery of CSF-1 binding activity by PEM even after prolonged incubation (7 days). IFN also inhibited the receptor-mediated uptake and utilization of CSF-1 molecules by treated cells, which appeared to be a direct effect of the decreased number of CSF-1 receptors. Treatment of PEM with dexamethasone, prostaglandin, transferrin, insulin, or dibutyryl cAMP failed to suppress both the expression of CSF-1 receptors and CSF-1 utilization by PEM. These studies suggest that IFN may play a role in the regulation of both macrophage production and differentiation via the modulation of specific membrane receptors and inhibition of receptor-mediated CSF-1 endocytosis.     

Distribution of cells bearing receptors for a colony-stimulating factor (CSF-1) in murine tissues

CSF-1 is a subclass of the colony-stimulating factors that specifically stimulates the growth of mononuclear phagocytes. We used the binding of 125I-CSF-1 at 0 degrees C by single cell suspensions from various murine tissues, in conjunction with radioautography, to determine the frequency of binding cells, their identity, and the number of binding sites per binding cell. For all tissues examined, saturation of binding sites was achieved within 2 h at 2--3 x 10(-10) M 125I-CSF-1. The binding was irreversible and almost completely blocked by a 2 h preincubation with 5 x 10(-10) M CSF-1. 125I-CSF-1 binding was exhibited by 4.3% of bone marrow cells, 7.5% of blood mononuclear cells, 2.4% of spleen cells, 20.5% of peritoneal cells, 11.8% of pulmonary alveolar cells and 0.4% of lymph node cells. Four morphologically distinguishable cell types bound 125I-CSF-1: blast cells; mononuclear cells with a ratio of nuclear to cytoplasmic area (N/C) greater than 1; cells with indented nuclei; and mononuclear cells with N/C less than or equal to 1. No CSF-1 binding cells were detected among blood granulocytes or thymus cells. Bone marrow promyelocytes, myelocytes, neutrophilic granulocytes, eosinophilic granulocytes, nucleated erythroid cells, enucleated erythrocytes, and megakaryocytes also failed to bind. The frequency distribution of grain counts per cell for blood mononuclear cells was homogenous. In contrast, those for bone marrow, spleen, alveolar, and peritoneal cells were heterogeneous. The monocytes in blood or bone marrow (small cells, with either indented nuclei or with N/C greater than 1) were relatively uniformly labeled, possessing approximately 3,000 binding sites per cell. Larger binding cells (e.g., alveolar cells) may possess higher numbers of receptors. It is concluded that CSF-1 binding is restricted to mononuclear phagocytic cells and their precursors and that it can be used to identify both mature and immature cells of this series.     

Solubilization and assay of a colony-stimulating factor receptor from murine macrophages

The colony-stimulating factor, CSF-1, selectively stimulates the survival, proliferation, and differentiation of mononuclear phagocytes. The solubilization, assay, and characteristics of the CSF-1 receptor from the J774.2 murine macrophage cell line are described. The recovery of cell-surface receptor in the postnuclear supernatant membrane fraction of hypotonically disrupted cells was 76%. Recovery of the ligand binding activity of the receptor after solubilization of this fraction with 1% Triton X-100 was approximately 150%. The binding of 125I-CSF-1 to intact cells and membrane preparations was consistent with the existence of a single class of high-affinity receptor sites. In contrast, the equilibrium binding of 125I-CSF-1 to the solubilized postnuclear fraction indicated the existence of two distinct classes of binding site (apparent Kds 0.15 nM and 10 nM). A rapid assay was developed for the high-affinity sites, which were shown to be associated with the CSF-1 receptor. The function of the low-affinity sites, which have not been demonstrated on intact cells or cell membranes and which are 13 times more abundant than the high-affinity sites, is unknown. The solubilized high-affinity receptor-CSF-1 complex was stable on storage at 0 degrees C and -70 degrees C but dissociated at 37 degrees C. Dissociation also occurred at 0 degrees C in buffers of low pH (4.0) or high ionic strength (0.7 M NaCl).     

Alterations in the expression of colony-stimulating factor-1 and its receptor during an acute graft-vs-host reaction in mice

During the course of an acute graft-vs-host reaction in the mouse we observed a progressive increase in the concentration of CSF-1 in serum and liver, peaking at day 14. In contrast, there was a progressive decrease in the splenic CSF-1 concentration. In vivo studies of 125I-CSF-1 uptake and degradation and in vitro studies of 125I-CSF-1 binding by splenic cells demonstrated that within 24 h of the reaction the number of CSF-1 receptor+ cells had increased by 2-fold and their capacity to express the CSF-1 receptor by approximately 3-fold, resulting in a approximately 2.5-fold increase in the splenic clearance of CSF-1 from the circulation. Inasmuch as at 24 h, serum CSF-1 was not significantly altered, these results are suggestive of an increased rate of release of CSF-1 into the circulation early in the response. The splenic CSF-1-receptor bearing cells were in a Mac-1+ fraction that is consistent with a role for CSF-1 in the generation of host-derived splenic macrophages in acute graft-vs-host reaction.     

Interleukin 3 (IL 3) regulates the in vitro proliferation of both blood monocytes and peritoneal exudate macrophages: synergism between a macrophage lineage-specific colony-stimulating factor (CSF-1) and IL 3

The effect of interleukin 3 (IL 3) on regulation of macrophage proliferation was examined. Although IL 3 alone stimulates the colony formation in bone marrow cells, it fails to stimulate the colony formation by both peritoneal exudate macrophages (PEM) and blood monocytes. However, IL 3 greatly enhances the proliferative capacity of both PEM and monocytes in responding to suboptimal concentrations of CSF-1. At supraoptimal concentrations of CSF-1, IL 3 did not increase the number of colonies, but greatly increased colony size. Kinetic studies showed that IL 3 enhances CSF-1-induced macrophage proliferation by shortening the cell doubling time. Monocytes were more sensitive to the action of IL 3 and possessed higher proliferative potential than PEM. Binding studies with radioactive labeled CSF-1 (125I-CSF-1) showed that IL 3 treatment induced an increased expression of CSF-1 receptor activity by PEM which appears to be a result of increased number of available receptor sites. The effect of IL 3 on the expression of receptor activity is both dose- and time-dependent. IL 3 also augments the rate of receptor-mediated CSF-1 endocytosis by PEM which appears to be a direct result of increased expression of CSF-1 binding sites. These results demonstrate that, in addition to stimulating the growth and differentiation of several blood cell lineages by hemopoietic stem cells, IL 3 also possesses the ability to modulate CSF-1 receptors, thereby affecting proliferation of more mature blood monocytes and tissue-derived macrophages.     

IL-1 and TNF transmodulate epidermal growth factor receptors by a protein kinase C-independent mechanism

The effect of the human rIL-1 alpha and rTNF-alpha on the binding of 125I-labeled epidermal growth factor ([125I]EGF) to its receptor (EGF-R) has been studied in human gingival fibroblasts (HuGi). Incubation of these cells with recombinant cytokines at 37 degrees C caused a rapid, dose-dependent decrease in their ability to subsequently bind subsaturating levels of [125I]EGF at 4 degrees C. Inhibition was evident at 5 min after addition of cytokines, reached a maximal level (60-70% reduction) after 15 to 30 min, and declined thereafter. Normal EGF binding was attained by 2 h. Half-maximal inhibition of EGF binding occurred at 10 pM IL-1 and 50 pM TNF. The two cytokines were not additive in their effect. Competition experiments at 4 degrees C showed that the cytokines did not interact directly with EGF-R; Scatchard analysis of binding of [125I]EGF to HuGi after treatment with IL-1 and TNF revealed an increase in EGF-R Kd from 0.75 nM to 2.9 nM with no change in receptor number. The effect of IL-1 and TNF on EGF-R was compared with that of the tumor-promotor PMA which is known to "transmodulate" EGF-R affinity by activating protein kinase C which then phosphorylates EGF-R. PMA caused a greater inhibition of EGF binding to HuGi (80 to 85% inhibition; ED50 = 500 pM), and recovery of binding was much slower. Importantly, in HuGi made deficient in protein kinase C by prolonged incubation with PMA, addition of fresh PMA no longer affected EGF binding, while the response to IL-1 and TNF was intact. Cytokine- but not PMA-mediated EGF-R transmodulation was partially reversed by treatment of the cells with millimolar concentrations of the kinase inhibitor amiloride. HuGi were incubated with H3 32PO4, stimulated with PMA or cytokines, and EGF-R were immunoprecipitated; IL-1 and TNF, like PMA, caused a 2- to 5-fold increase in receptor phosphorylation. We conclude that occupation of IL-1 and TNF-R activates a protein kinase, distinct from kinase C, for which EGF-R is a substrate.     

Interaction of murine colony-stimulating factor (CSF-1) with alveolar mononuclear phagocytes

Colony-stimulating factor (CSF-1) was purified from serum-free L-cell-conditioned medium (LCM) and iodinated so that we could study its interaction with murine alveolar macrophages. At 0 °C, the binding of ¹²⁵ICSF-1 to alveolar macrophages reached a stable maximum within 16 h. Under this condition, the binding of ¹²⁵ICSF-1 at various concentrations was saturated at about 3 ng/ml. The binding sites of ¹²⁵ICSF-1 were sensitive to trypsin but not to DNase or RNase treatment. At 37 °C, the trypsin-treated cells regenerated more than 90% of their original binding sites within 12 h. Whereas more than 97% of these alveolar macrophages were phagocytic and esterase-positive, autoradiographic studies showed that only 10–31 % of them were capable of binding to ¹²⁵ICSF-1. These results indicate that the frequencies of CSF-1-binding cells and alveolar macrophage colony-forming cells (AL-CFC) are closely correlated, but no causal relationship has been established.     

The specific binding activities of human urinary radioiodinated colony-stimulating factor-1 to various human tissue cells

1. Colony-stimulating factor (CSF-1) was isolated from a large volume of fresh normal human urine by 5 steps of purification and enrichment. 2. The purification factor is 100,000 fold and the purified compound exhibits a 2.16 x 10(7) U/mg of protein sp. act. 3. The isolated CSF-1 is a sialoglycoprotein with 41.5% of carbohydrate. The almost complete removal of this carbohydrate moiety (up to 91%) was achieved by incubation with trifluoromethane sulfonic acid. 4. The deglycosylated CSF-1 (DG-CSF-1) possesses an apparent Mr 38,000 compared to native CSF-1 with an initial Mr 57,000 (Goa et al., 1988). 5. The features of the interaction of radio-iodinated [125I]CSF-1 with single cell suspensions from various human tissues (bone marrow, spleen, blood, peritoneal cavity, alveolar lavage, lymph node and thymus), were studied. 6. The binding activity of peritoneal macrophages was the highest among the cells examined and erythrocytes, thymus and blood granulocytes showed no CSF-1 binding. 7. On incubation with [125I]CSF-1 at 0 degrees C, cellular binding of [125I]CSF-1 reached a stable maximum within 16 hr. This is in contrast to the association behaviour at higher temperature. 8. At 37 degrees C, cellular associated [125I]CSF-1 levels reached, within 90 min, an unstable maximum which was up to 10 times less than that occurring under the same conditions at 0 degree C. From the Scatchard plot analysis, we obtained the affinity constant and the number of receptor(s). 9. The binding site is sensitive to trypsin. 10. The receptor alone, (labelled by cross-linking to [125I]CSF-1 with di-succinylimidyl-suberate), is a polypeptide with an approx. Mr 110,000. 11. Our results showed that the receptor of CSF-1 is a tyrosin-kinase.     

Colony-stimulating factor-1 modulates alpha 2-macroglobulin receptor expression in murine bone marrow macrophages.

Primary cultures of murine bone marrow macrophages (BMMs) were prepared from marrow cell suspensions. These cells expressed specific receptors that recognized the transformed conformation of human alpha 2-macroglobulin (alpha 2M) generated by reaction with CH3NH2. alpha 2M receptor expression was regulated by colony-stimulating factor-1 (CSF-1). The BMMs were deprived of CSF-1 for 6 h and then treated with different concentrations of the purified cytokine. After 18 h, binding of 125I-alpha 2M-CH3NH2 was examined at 4 degrees C. Analysis of the saturation isotherms and Scatchard transformations indicated that the KD was not affected by CSF-1 (1.9-2.4 nM), whereas the maximum specific radioligand binding capacity (Bmax) was increased from 5.6 x 10(4) receptors/cell in the absence of CSF-1 to 2.2 x 10(5) and 2.6 x 10(5) receptors/cell for BMMs treated with 1,000 and 10,000 units/ml CSF-1, respectively. The difference in total cellular protein after exposure to different levels of CSF-1 for 18 h was small (1.50-1.92 ng/cell) and not statistically significant. A 6-12-h lag phase was identified between the time of CSF-1 exposure and increased alpha 2M receptor expression. Cycloheximide completely blocked the increase in alpha 2M receptor expression when added simultaneously with the CSF-1; greater than 50% inhibition was observed when the cycloheximide was added up to 8 h later. The RNA synthesis inhibitors, actinomycin D and daunomycin, prevented increased alpha 2M receptor expression when added up to 4 h after the CSF-1, but had no effect at 8 h. At 37 degrees C, uptake and digestion of 125I-alpha 2M-CH3NH2 was increased in BMMs treated with 1,000 units/ml CSF-1 for 18 h compared with untreated cells. These studies demonstrate that CSF-1 increases the expression of alpha 2M receptors in BMMs through a pathway that requires new RNA and protein synthesis. We hypothesize that increased alpha 2M receptor expression may play an important role in cellular growth and differentiation.     

Lineage specific receptors used to identify a growth factor for developmentally early hemopoietic cells: assay of hemopoietin-2

A new approach, based on the occurrence of receptors for the mononuclear phagocyte lineage specific hemopoietic growth factor (HGF) colony stimulating factor-1 (CSF-1) on developmentally early multipotent cells, is utilized to detect and assay rapidly another HGF, hemopoietin-2. This method is also used to determine the relative maturity of hemopoietin-2 target cells, to investigate synergism between hemopoietin-2 and CSF-1, and to measure CSF-1 receptor levels on maturing cells. While the target cell specificities of hemopoietin-2 and CSF-1 overlap, hemopoietin-2 causes the appearance of developmentally earlier 125I-CSF-1 binding cells de novo in the absence of CSF-1. Increased CSF-1 receptor densities are observed on cells incubated with either HGF, consistent with acquisition of the capacity for increased expression of the receptor by mononuclear phagocyte progenitor cells just prior to their differentiation to adherent mononuclear phagocytes. Together, both HGFs have a synergistic effect on the generation of 125I-CSF-1 binding cells with elevated CSF-1 receptor densities. Preliminary characterization of hemopoietin-2 from medium conditioned by WEHI-3 cells indicates that it is very similar to, if not identical with, interleukin-3 (IL-3) and the HGF(s) acting on multipotential cells and cells giving rise to erythroid cells, granulocytes, mononuclear phagocytes, and megakaryocytes. Purified IL-3 was shown to possess hemopoietin-2 activity.