Role of granulocyte/macrophage colony-stimulating factor in the regulation of murine alveolar macrophage proliferation and differentiation

Granulocyte/macrophage (GM)-CSF is one of the hemopoietic growth factors that stimulates neutrophilic granulocyte and macrophage production by bone marrow progenitor cells. In this study, the effect of GM-CSF on the growth and differentiation of murine pulmonary alveolar macrophages (PAM) was investigated. In the presence of GM-CSF, normal murine PAM were induced to proliferate and develop into macrophage colonies with a dose-response curve similar to that of bone marrow GM colony-forming cells. PAM also responded to CSF-1, a lineage-restricted growth factor, but required much higher doses of CSF-1 and a longer incubation time for optimal colony formation. The proliferative response of PAM to CSF-1, however, was greatly enhanced by the concurrent addition of low doses of GM-CSF. In contrast, low doses of CSF-1 failed to potentiate the proliferative response of PAM to GM-CSF. Macrophages derived from GM-CSF cultures were rounder and less stretched and possessed less FcR-mediated phagocytic activity than cells produced in CSF-1 cultures. A study with hydrocortisone-induced monocytopenia showed that nearly one half of lung macrophages may be sustained by local proliferation of PAM without the continuous migration of blood monocytes. This study suggests that GM-CSF may play a major role in the production of PAM by two modes of action, 1) direct stimulation of cell proliferation and 2) enhancement of their responsiveness to CSF-1, thereby producing more mature and functionally competent macrophages.     

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.     

Effects of granulocyte-macrophage colony-stimulating factor and colony-stimulating factor-1 on the proliferation and differentiation of murine alveolar macrophages

Murine alveolar macrophages (AM) were shown to have proliferative ability and to form colonies in vitro. The factors in lung-conditioned medium (CM) and L929-CM which stimulate the proliferation of AM were considered to be granulocyte-macrophage colony-stimulating factor (GM-CSF) and CSF-1, respectively, because recombinant murine (rm)GM-CSF and recombinant human (rh)CSF-1 could replace the activities of lung-CM and L929-CM, respectively. The phenotype of the cells in the colonies formed by AM incubated with rmGM-CSF or lung-CM was AM-like; more than 90% of the cells were stained by anti-asialo GM1 but not by FITC-LPS, and had AM-like morphology. Expression of Mac-1 Ag determined by M1/70HL in these cells as well as original AM was low. However, the phenotype of the cells in the colonies formed by AM incubated with rhCSF-1 or L929-CM was peritoneal macrophage (PM)-like; more than 90% of the cells were stained by FITC-LPS and M1/70HL, but not by anti-asialo GM1, and showed PM-like morphology. The cells in the colonies formed by AM incubated with rmGMCSF changed their phenotype after treatment with rhCSF-1; the percentage of cells stained by anti-asialo GM1 decreased, and that of cells stained by FITC-LPS increased. The cells in the colonies formed by AM incubated with rhCSF-1 never changed their phenotype after incubation with rmGM-CSF. In contrast to AM, more than 90% of the cells in all colonies formed by PM incubated with either rmGM-CSF, rhCSF-1, lung-CM, or L929-CM were stained by FITC-LPS but not by anti-asialo GM1. These results show that although AM and PM can proliferate, AM, in contrast to PM, are bipotential cells that can differentiate into two types of macrophages responding to distinct types of CSF, and that one of the molecular mechanisms controlling macrophage heterogeneity may be based on the type of CSF produced at distinct tissues.     

The nature of 12-O-tetradecanoylphorbol-13-acetate (TPA)-stimulated hemopoiesis, colony stimulating factor (CSF) requirement for colony formation, and the effect of TPA on [125I]CSF-1 binding to macrophages

The tumor-promoting phorbol diester, 12-O-tetradecanoylphorbol-13-acetate (TPA) was found to act both independently of and synergistically with the mononuclear phagocyte specific colony stimulating factor (CSF-1) to stimulate the formation of macrophage colonies in cultures of mouse bone marrow cells. In contrast, TPA did not synergize with other CSF subclasses that stimulate the formation of eosinophil, eosinophil-neutrophil, neutrophil, neutrophil-macrophage, and macrophage colonies, nor with either of the two factors required for megakaryocyte colony formation, megakaryocyte CSF, and megakaryocyte colony potentiator. In serum-free mouse bone marrow cell cultures TPA retained the ability to independently stimulate macrophage colony formation. However, TPA-stimulated colony formation was suboptimal and delayed in serum-free cultures that could support optimal colony formation in the presence of CSF-1. In addition, TPA did not directly compete with [125I]CSF-1 at 4 degrees C for its specific, high-affinity receptor on mouse peritoneal exudate macrophages. However, a 2-hour preincubation of the cells with TPA at 37 degrees caused almost complete loss of the receptor. Thus, TPA is able to mimic CSF-1 in its effects on CSF-1 responsive cells in some aspects (the spectrum of target cells, the morphology of resulting colonies, and the ability to down-regulate the CSF-1 receptor) but it is not able to mimic CSF-1 in other ways (TPA alone cannot stimulate the full CSF-1 response, TPA does not stimulate the most primitive CSF-1 responsive cells, and TPA does not bind to the CSF-1 receptor).     

Interactions between purified GM-CSF, purified erythropoietin and spleen conditioned medium on hemopoietic colony formation in vitro.

Preincubation of C57BL adult marrow cells or CBA fetal liver cells with a 250-fold excess concentration of purified GM-CSF failed to reduce the frequency of cells forming eosinophil, megakaryocyte or erythroid colonies in subsequent agar cultures. When excess concentrations of purified GM-CSF were added to agar cultures stimulated by pokeweed mitogen-stimulated spleen conditioned medium (SCM), no reduction was observed in the frequency of eosinophil, megakaryocyte or erythroid colonies. Addition of 4 units of purified erythropoietin (EPO) to cultures of fetal liver or adult marrow cells stimulated by SCM increased the number of erythroid colonies but did not reduce the number of non-erythroid colonies or the non-erythroid content of mixed erythroid colonies. Although neither GM-CSF nor EPO alone was able to stimulate erythroid colony formation in agar cultures of fetal liver cells, small numbers of large erythroid colonies were stimulated to develop in cultures containing both purified regulators. Purified GM-CSF was also able to support the survival in vitro of a small proportion of erythroid colony-forming cells in fetal liver populations cultured initially in the absence of SCM and the survival of some eosinophil and megakaryocyte colony-forming cells in similar cultures of adult marrow cells. The results do not support the hypothesis that GM-CSF and EPO compete for a common pool of uncommitted progenitor cells. On the contrary, the data indicate that GM-CSF und EPO are able to collaborate in stimulating the proliferation of some erythropoietic cells. Furthermore, purified GM-CSF appears to be able to support temporarily the survival and/or initial proliferation of at least some cells forming erythroid, eosinophil and megakaryocyte colonies, even though GM-CSF is unable to stimulate the formation of colonies of these types.     

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.     

Hierarchical down-modulation of hemopoietic growth factor receptors

Granulocytes and macrophages can be produced in vitro when progenitor cells from mouse bone marrow are stimulated by any of four distinct colony stimulating factors, Multi-CSF (IL-3), GM-CSF, G-CSF, and M-CSF (CSF-1). At 0 degrees C the four CSFs do not cross-compete for binding to bone marrow cells, indicating that each has a specific cell surface receptor. However, at 21 degrees C or 37 degrees C, Multi-CSF inhibits binding of the other three CSFs and GM-CSF inhibits binding of G-CSF and M-CSF. Rather than competing directly for receptor binding, the binding of Multi-CSF, GM-CSF, or G-CSF to their own receptor induces the down-modulation (and thus activation) of other CSF receptors at 37 degrees C. The pattern and potency of down-modulation activity exhibited by each type of CSF parallels the pattern and potency of its biological activity. We propose a model in which the biological interactions of the four CSFs are explained by their ability to down-modulate and activate lineage-specific receptors.     

Modulation of colony-stimulating factor-1 receptors on macrophages by tumor necrosis factor

The effect of murine rTNF-alpha on the binding of human 125I-rCSF-1 to murine thioglycolate-elicited peritoneal exudate macrophages (PEM) was investigated. At 4 degrees C, 125I-CSF-1 binding to PEM was inhibited by preincubation with human rCSF-1, but not by other cytokines. When PEM were incubated with various cytokines at 37 degrees C, murine rTNF-alpha caused greater than 90% decrease in 125I-CSF-1 binding. This decrease was time, temperature and TNF dose dependent, and was not affected by preincubation with cycloheximide. The reduction in CSF-1-binding activity was reversed by prolonged incubation at 37 degrees C even in the presence of TNF. However, PEM preincubated with TNF subsequently washing free of residual TNF resulted in a rapid recovery of CSF-1 binding. This recovery of CSF-1-binding activity required protein synthesis. Binding studies suggested that the decrease in 125I-CSF-1 binding was most likely caused by a reduction in the number of CSF-1 receptors. In addition, preincubation with TNF at 37 degrees C inhibited 125I-CSF-1 binding on mononuclear phagocytes, including the macrophage cell line J774, bone marrow-derived macrophages, and nonelicited macrophages from three different strains of mice. In contrast, 125I-murine rTNF-alpha binding to PEM was not inhibited by preincubation with CSF-1 at 4 degrees C or 37 degrees C. These data suggest that TNF may play a role in the modulation of receptor expression on blood cells, and may point to a role for this pleiotropic cytokine in the regulation of hemopoiesis.     

The role of calcitonin gene-related peptide (CGRP) in macrophages: the presence of functional receptors and effects on proliferation and differentiation into osteoclast-like cells

It has been shown that both calcitonin gene-related peptide (CGRP) and amylin bind weakly to calcitonin (CT) receptors in osteoclast-like cells formed in vitro and inhibit bone resorption by a cAMP-dependent mechanism. Osteoclasts are thought to be derived from cells of the monocyte macrophage lineage, in which CGRP, but not CT, induces cAMP production. In this study, we determined the presence of functional receptors for CGRP in mouse alveolar macrophages and the effects of this peptide on proliferation and osteoclastic differentiation in mouse alveolar and bone marrow-derived macrophages. Human CT did not stimulate cAMP production in macrophages. Human CGRP stimulated cAMP production in mouse alveolar macrophages and bone marrow-derived macrophages dose-dependently. Human amylin, which has 43% homology with human CGRP, also stimulated these macrophages to produce cAMP, but only at a 100-fold higher concentration. The increment in cAMP production induced by human CGRP and amylin was abolished by the addition of human CGRP(8–37), a selective antagonist for CGRP receptors. Specific binding of [¹²⁵I]human CGRP to alveolar macrophages was detected (dissociation constant, 2.5 × 10⁻⁸ M; binding sites, 1.4 × 10⁴/cell). Amylin, but not CT, displaced the bound [¹²⁵I]human CGRP from alveolar macrophages, but at a 100-fold higher concentration. No specific binding of [¹²⁵I]human CT and [¹²⁵I]human amylin to alveolar macrophages could be detected. Pretreatment with human CGRP for 24 h dose-dependently suppressed DNA synthesis in alveolar macrophages induced by granulocyte-macrophage colony-stimulating factor (GM-CSF). CGRP also suppressed the number of macrophage colonies formed from bone marrow cells induced by macrophage colony-stimulating factor (M-CSF). Pre-treatment of alveolar macrophages with CGRP inhibited differentiation into osteoclast-like cells in co-cultures with primary osteoblastic cells in the presence of 1α,25-dihydroxy vitamin D₃. These results indicate that specific receptors for CGRP are present in macrophages and that CGRP modulates proliferation and differentiation of macrophages into osteoclast-like cells by a receptor-mediated mechanism involving cAMP.     

Control of macrophage lineage populations by CSF-1 receptor and GM-CSF in homeostasis and inflammation

There is recent interest in the role of monocyte/macrophage subpopulations in pathology. How the hemopoietic growth factors, macrophage-colony stimulating factor (M-CSF or CSF-1) and granulocyte macrophage (GM)-CSF, regulate their in vivo development and function is unclear. A comparison is made here on the effect of CSF-1 receptor (CSF-1R) and GM-CSF blockade/depletion on such subpopulations, both in the steady state and during inflammation. In the steady state, administration of neutralizing anti-CSF-1R monoclonal antibody (mAb) rapidly (within 3-4 days) lowered, specifically, the number of the more mature Ly6C(lo) peripheral blood murine monocyte population and resident peritoneal macrophages; it also reduced the accumulation of murine exudate (Ly6C(lo)) macrophages in two peritonitis models and alveolar macrophages in lung inflammation, consistent with a non-redundant role for CSF-1 (or interleukin-34) in certain inflammatory reactions. A neutralizing mAb to GM-CSF also reduced inflammatory macrophage numbers during antigen-induced peritonitis and lung inflammation. In GM-CSF gene-deficient mice, a detailed kinetic analysis of monocyte/macrophage and neutrophil dynamics in antigen-induced peritonitis suggested that GM-CSF was acting, in part, systemically to maintain the inflammatory reaction. A model is proposed in which CSF-1R signaling controls the development of the macrophage lineage at a relatively late stage under steady state conditions and during certain inflammatory reactions, whereas in inflammation, GM-CSF can be required to maintain the response by contributing to the prolonged extravasation of immature monocytes and neutrophils. A correlation has been observed between macrophage numbers and the severity of certain inflammatory conditions, and it could be that CSF-1 and GM-CSF contribute to the control of these numbers in the ways proposed.     

Interactions among granulocyte-macrophage colony-stimulating factor, macrophage colony-stimulating factor, and IFN-gamma lead to enhanced proliferation of murine macrophage progenitor cells

This report examines the actions of IFN-gamma on monocytopoiesis in murine liquid and semisolid bone marrow cultures. The proliferative response of bone marrow cells to macrophage CSF and granulocyte-macrophage CSF was assayed by measuring [3H]TdR uptake in a range of mouse strains. No interstrain difference in kinetics was observed for CSF-1 action, but GM-CSF acted significantly more rapidly on C57B1/6, Swiss, and to a lesser extent A/J mice than on BALB/c or CBA. IFN-gamma inhibited [3H]TdR incorporation elicited by CSF-1, and to a much lesser extent, GM-CSF. When the two CSF were added together, the effects were not additive; in fact, the response was the same as that seen with GM-CSF alone. When IFN-gamma was also added, the response was restored to the level seen with CSF-1 alone. In essence, the inhibitory actions of GM-CSF and IFN-gamma were mutually exclusive. The mechanism of these actions was investigated using colony assays. As expected, CSF-1 caused the formation of pure macrophage colonies, whereas GM-CSF stimulated production of macrophage, granulocyte, and mixed granulocyte macrophage colonies. When the two CSF were added in combination, the total colony count was greater than with either alone, but less than additive. The number of pure macrophage colonies was reduced to the number seen with GM-CSF alone. IFN-gamma reduced the number of colonies in the presence of CSF-1, but slightly increased the number with GM-CSF. In the presence of both CSF, IFN-gamma increased the colony count by around 25 to 40%, so that the numbers were greater than the combined total of CSF-1 plus GM-CSF added separately. Similar results were obtained in all mouse strains tested. The results suggest that the thymidine uptake data reflect changes in the number of progenitor cells responding rather than changes in cell cycle time. The results are discussed in terms of the possibility that coadministration of GM-CSF and CSF-1 could ameliorate the myelosuppressive actions of IFN-gamma in vivo, leading to more effective use of this agent as a biologic response modifier.     

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.     

The regulation of mononuclear phagocyte entry into S phase by the colony stimulating factor CSF-1

CSF-1 is a hemopoietic growth factor that specifically regulates the survival, proliferation, and differentiation of mononuclear phagocytic cells. Populations of adherent bone marrow-derived macrophages (BMM) devoid of CSF-1 producing cells were used to study regulation by CSF-1 of macrophage entry into S phase. More than 95% of BMM possess the CSF-1 receptor. It was shown that 93-98% of BMM are cycling (S phase 8-9 hr, doubling time 24-28 hr) when cultured in the presence of CSF-1. BMM incubated with 15% FCS in the absence of CSF-1 or in the presence of CSF-1 concentrations inducing survival without proliferation enter a quiescent state. This state is characterized by a reduction in the synthesis of DNA (98%), total protein (35%), ribosomal protein (76%), and histone (96%) compared with the synthetic rate of these components in exponentially growing cells. Addition of CSF-1 to BMM rendered quiescent by removal of CSF-1 stimulated entry into S phase with a lag period of approximately 12 h. This lag period is reduced to 8 hr in BMM made quiescent at concentrations of CSF-1 inducing survival without proliferation, an effect which may be related to the expected higher protein content of these cells (Tushinski and Stanley, J. Cell. Physiol., 116:67-75). Neutralization of CSF-1 by antibody at different times during the lag period indicates that CSF-1 is required for almost the entire lag period for the entry of any cells into S phase. In BMM rendered quiescent by removal of both serum and CSF-1, purified CSF-1 without serum stimulated entry of cells into S phase, whereas serum alone was ineffective. The results are consistent with a primary regulatory role of CSF-1 in mononuclear phagocyte proliferation, survival, and function.     

ERK5/BMK1 is indispensable for optimal colony-stimulating factor 1 (CSF-1)-induced proliferation in macrophages in a Src-dependent fashion

CSF-1, by binding to its high-affinity receptor CSF-1R, sustains the survival and proliferation of monocyte/macrophages, which are central cells of innate immunity and inflammation. The MAPK ERK5 (also known as big MAPK-1, BMK1, or MAPK7) is a 98-kDa molecule sharing high homology with ERK1/2. ERK5 is activated by oxidative stress or growth factor stimulation. This study was undertaken to characterize ERK5 involvement in macrophage signaling that is elicited by CSF-1. Exposure to the CSF-1 of primary human macrophages or murine macrophage cell lines, as well as murine fibroblasts expressing ectopic CSF-1R, resulted in a rapid and sustained increase of ERK5 phosphorylation on activation-specific residues. In the BAC1.2F5 macrophage cell line, ERK5 was also activated by another mitogen, GM-CSF, while macrophage activators such as LPS or IFN-gamma and a number of nonproliferative cytokines failed. Src family kinases were found to link the activation of CSF-1R to that of ERK5, whereas protein kinase C or the serine phosphatases PP1 and PP2A seem not to be involved in the process. Treatment of macrophages with ERK5-specific small interfering RNA markedly reduced CSF-1-induced DNA synthesis and total c-Jun phosphorylation and expression, while increasing the expression of the cyclin-dependent kinase inhibitor p27. Following CSF-1 treatment, the active form of ERK5 rapidly translocated from cytosol to nucleus. Taken together, the results reported in this study show that ERK5 is indispensable for optimal CSF-1-induced proliferation and indicate a novel target for its control.     

Relative levels of M-CSF and GM-CSF influence the specific generation of macrophage populations during infection with Mycobacterium tuberculosis

Members of the CSF cytokine family play important roles in macrophage recruitment and activation. However, the role of M-CSF in pulmonary infection with Mycobacterium tuberculosis is not clear. In this study, we show the lungs of mice infected with M. tuberculosis displayed a progressive decrease in M-CSF in contrast to increasing levels of GM-CSF. Restoring pulmonary M-CSF levels during infection resulted in a significant decrease in the presence of foamy macrophages and increased expression of CCR7 and MHC class II, specifically on alveolar macrophages. In response to M-CSF, alveolar macrophages also increased their T cell-stimulating capacity and expression of DEC-205. These studies show that the levels of expression of M-CSF and GM-CSF participate in the progression of macrophages into foamy cells and that these cytokines are important factors in the differentiation and regulation of expression of dendritic cell-associated markers on alveolar macrophages. In addition, these studies demonstrate that M-CSF may have a role in the adaptive immune response to infection with M. tuberculosis.     

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.     

Growth factors of lower vertebrates: characterization of goldfish (Carassius auratus L.) macrophage colony-stimulating factor-1

Colony-stimulating factor-1 (CSF-1) regulates mononuclear cell proliferation, differentiation, and survival. The functions of CSF-1 are well documented in mammals; however, little is known about CSF-1 biology in lower vertebrates. This is the first report on the identification and functional characterization of a fish CSF-1 molecule expressed highly in the spleen and in phorbol 12-myristate 13-acetate-stimulated monocytes. Goldfish CSF-1 is a 199-amino acid protein that possesses the required cysteine residues to form important intra-chain and inter-chain disulfide bonds that allow CSF-1 to form a functional homodimer and to interact with its high affinity receptor, CSF-1R. Recombinant goldfish CSF-1 formed a homodimer and bound to the soluble goldfish CSF-1R. The addition of the recombinant CSF-1 to sorted goldfish progenitor cells, monocytes, and macrophages induced the differentiation of monocytes into macrophages and the proliferation of monocyte-like cells. The proliferation of these cells was abrogated by addition of an anti-CSF-1R antibody as well as the soluble CSF-1R. The ability of the soluble CSF-1R to inhibit CSF-1-induced proliferation represents a novel mechanism for the regulation of CSF-1 function.     

Stimulation of macrophage tumoricidal activity by the growth and differentiation factor CSF-1

The effect of the macrophage growth and differentiation factor CSF-1 on the tumoricidal capacity of murine peritoneal exudate macrophages was investigated. Pretreatment of peptone-elicited macrophages 1 day with 300-1200 U/ml CSF-1 induced moderate killing and greatly stimulated lymphokine (LK)-induced killing of [3H]thymidine-labeled TU5 sarcoma cells to levels above that seen with fresh macrophages. Further addition of CSF-1 at Day 1 at the time of the tumor lysis assay promoted moderate increases in spontaneous and LK-induced activity. CSF-1 did not stimulate freshly harvested exudate macrophages to lyse TU5 targets in the presence or absence of lymphokine (LK) activators. Lipopolysaccharide (LPS) at 0.1-1000 ng/ml did not stimulate cytotoxicity, and the low endotoxin content and the use of polymyxin B and C3H/HeJ mice excluded a role for LPS in these experiments. Incubation of the macrophages with IFN and the myeloid growth factors IL-3 and GM-CSF did not stimulate tumoricidal activity. CSF-1 has been proposed as a therapeutic agent to restore myeloid cell numbers in induced (cancer chemotherapy, bone marrow transplantation, etc.) and natural aplastic anemias. These studies show that CSF-1 also may be useful in combination with LK activators to promote resistance to cancer in mature mononuclear cells. CSF-1 may have similar effects in LK-activated macrophages to enhance resistance to infectious diseases.     

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.     

Regulation of hemopoietic cell differentiation and proliferation

Differentiation and proliferation of almost all hemopoietic cell lines can now be studied in vitro. Cloning techniques and suspension cultures allow the study of proliferation of the multipotential hemopoietic progenitor cell and the committed progenitors for granulocytes, macrophages, eosinophils, megakryocytes, and erythrocytes. The proliferation of each of the committed progenitor cells is controlled by specific glycoproteins and two of these have recently been purified: granulocyte-macrophage colony-stimulating factor (GM-CSF) and erythropoietin. The rate of proliferation of the GM-progenitor cells and their pattern of differentiation depends on the concentration of the hormone. At low concentrations of GM-CSF (10^?11 M) fewer progenitor cells are stimulated and macrophage colonies rather than granulocyte colonies develop. The change in the direction of granulocyte-macrophage differentiation appears to be related to (a) the concentration of GM- CSF and (b) the different sensitivity of a subpopulation of monocyte colony-forming cells which are responsive to GM-CSF even at low concentrations of the regulator. Analysis of the rate of RNA synthesis by bone marrow cells has shown that GM-CSF stimulates the mature nondividing end cells of differentiation (ie, polymorphs) as well as the progenitor cells. Although GM-CSF and erythropoietin have been radiolabeled, binding studies have been hampered by the loss of biologic activity during the labeling procedure and the heterogeneity of the target cells to which the regulators bind. Surface proteins and receptors for erythrocytes have been well characterized but the relationships between these proteins and the cell surface proteins of nucleated blood cells is not well understood. It appears that some proteins are lost from the cell surface during the development of granulocytes, which are retained on the surface of the B lymphocyte. Other proteins such as chemotactic receptors and complement receptors only appear on the mature cells. External radiolabeling of the granulocyte surface using iodogen yielded a simple profile of ¹²⁵I-labeled proteins when analyzed by sodium dodecyl sulphate polyacrylamide gel electrophoresis.