Membrane-bound Macrophage Colony-stimulating Factor Research Articles

Specific targeting of PKC\u03b4 suppresses osteoclast differentiation by accelerating proteolysis of membrane-bound macrophage colony-stimulating factor receptor

c-Fms is the macrophage colony-stimulating factor (M-CSF) receptor, and intracellular signalling via the M-CSF/c-Fms axis mediates both innate immunity and bone remodelling. M-CSF-induced transient proteolytic degradation of c-Fms modulates various biological functions, and protein kinase C (PKC) signalling is activated during this proteolytic process via an unknown mechanism. Notably, the role of specific PKC isoforms involved in c-Fms degradation during osteoclast differentiation is not known. Here, we observed that inactivation of PKCδ by the biochemical inhibitor rottlerin, a cell permeable peptide inhibitor, and short hairpin (sh) RNA suppresses osteoclast differentiation triggered by treatment with M-CSF and receptor activator of NF-κB ligand. Interestingly, inhibition of PKCδ by either inhibitor or gene silencing of PKCδ accelerated M-CSF-induced proteolytic degradation of membrane-bound c-Fms via both the lysosomal pathway and regulated intramembrane proteolysis (RIPping), but did not affect c-fms expression at the mRNA level. Degradation of c-Fms induced by PKCδ inactivation subsequently inhibited M-CSF-induced osteoclastogenic signals, such as extracellular signal-regulated kinase (ERK), c-JUN N-terminal kinase (JNK), p38, and Akt. Furthermore, mice administered PKCδ inhibitors into the calvaria periosteum exhibited a decrease in both osteoclast formation on the calvarial bone surface and the calvarial bone marrow cavity, which reflects osteoclastic bone resorption activity. These data suggest that M-CSF-induced PKCδ activation maintains membrane-anchored c-Fms and allows the sequential cellular events of osteoclastogenic signalling, osteoclast formation, and osteoclastic bone resorption.

Heterogeneous effects of M-CSF isoforms on the progression of MLL-AF9 leukemia.

Macrophage colony-stimulating factor (M-CSF) regulates both malignant cells and microenvironmental cells. Its splicing isoforms show functional heterogeneity. However, their roles on leukemia have not been well established. Here, the expression of total M-CSF in patients with hematopoietic malignancies was analyzed. The roles of M-CSF isoforms on the progression of acute myeloid leukemia (AML) were studied by establishing MLL-AF9-induced mouse AML models with high level membrane-bound M-CSF (mM-CSF) or soluble M-CSF (sM-CSF). Total M-CSF was highly expressed in myeloid leukemia patients. Furthermore, mM-CSF but not sM-CSF prolonged the survival of leukemia mice. While sM-CSF was more potent to promote proliferation and self-renew, mM-CSF was more potent to promote differentiation. Moreover, isoforms had different effects on leukemia-associated macrophages (LAMs) though they both increase monocytes/macrophages by growth-promoting and recruitment effects. In addition, mM-CSF promoted specific phagocytosis of leukemia cells by LAMs. RNA-seq analysis revealed that mM-CSF enhanced phagocytosis-associated genes and activated oxidative phosphorylation and metabolism pathway. These results highlight heterogeneous effects of M-CSF isoforms on AML progression and the mechanisms of mM-CSF, that is, intrinsically promoting AML cell differentiation and extrinsically enhancing infiltration of macrophages and phagocytosis by macrophages, which may provide potential clues for clinical diagnosis and therapy.

Diverse in vivo effects of soluble and membrane-bound M-CSF on tumor-associated macrophages in lymphoma xenograft model.

Macrophage colony-stimulating factor (M-CSF) is an important cytokine for monocyte/macrophage lineage. Secretory M-CSF (sM-CSF) and membrane-bound M-CSF (mM-CSF) are two major alternative splicing isoforms. The functional diversity of these isoforms in the activation of tumor-associated macrophages (TAMs), especially in lymphoma microenvironment, has not been documented. Here, we studied the effects of M-CSF isoforms on TAMs in xenograft mouse model. More infiltrating TAMs were detected in microenvironment with mM-CSF and sM-CSF. TAMs could be divided into three subpopulations based on their expression of CD206 and Ly6C. While sM-CSF had greater potential to recruit and induce differentiation of TAMs and TAM subpopulations, mM-CSF had greater potential to induce proliferation of TAMs and TAM subpopulations. Though both isoforms educated TAMs and TAM subpopulations to M2-like macrophages, mM-CSF and sM-CSF induced different spectrums of phenotype-associated genes in TAMs and TAM subpopulations. These results suggested the diverse effects of M-CSF isoforms on the activation of TAMs and TAM subpopulations in lymphoma microenvironments.

Membrane-bound macrophage colony-stimulating factor mediated auto-juxtacrine downregulates matrix metalloproteinase-9 release on J6-1 leukemic cell.

Earlier studies indicate that J6-1 human leukemic cells proliferate and propagate via the membrane-bound macrophage colony-stimulating factor (M-CSF)-mediated auto-juxtacrine mechanism. Matrix metalloproteinases (MMPs) can modulate the activity of cell membrane molecules and influence many cellular behaviors. Therefore, we hypothesized that MMP may also be involved in the membrane-bound M-CSF-mediated juxtacrine mechanism. First, we investigated whether blocking of membrane-bound M-CSF by neutralizing antibody to M-CSF or M-CSF receptor and adding of exogenous M-CSF are able to influence MMP-9 release. Next, we determined whether MMP-9 participated in J6-1 cells proliferation and influence the shedding of membrane-bound M-CSF and its receptor. Current studies show that blockade of the interaction between membrane-bound M-CSF and M-CSF receptor by antibody to M-CSF or M-CSF receptor promotes MMP-9 release. Moreover, we demonstrated that because of M-CSF mediated juxtacrine, lack of MMP-9 promotes J6-1 cell proliferation, in which a decrease in the shedding of cell-surface M-CSFR is involved. Hence, we suggest that membrane-bound M-CSF inhibit MMP-9 release and down-regulated MMP-9 contribute to juxtacrine stimulating in leukemic cell growth.

Increasing membrane-bound MCSF does not enhance OPGL-driven osteoclastogenesis from marrow cells

Macrophage colony-stimulating factor (MCSF) and osteoprotegerin ligand (OPGL), both produced by osteoblasts/stromal cells, are essential factors for osteoclastogenesis. Whether local MCSF levels regulate the amount of osteoclast formation is unclear. Two culture systems, ST-2 and Chinese hamster ovary-membrane-bound MCSF (CHO-mMCSF)-Tet-OFF cells, were used to study the role of mMCSF in osteoclast formation. Cells from bone marrow (BMM) or spleen were cultured with soluble OPGL on glutaraldehyde-fixed cell layers; osteoclasts formed after 7 days. Osteoclast number was proportional to the amount of soluble OPGL added. In contrast, varying mMCSF levels in the ST-2 or CHO-mMCSF-Tet-OFF cell layers, respectively by variable plating or by addition of doxycycline, did not affect BMM osteoclastogenesis: 20-450 U of mMCSF per well generated similar osteoclast numbers. In contrast, spleen cells were resistant to mMCSF: osteoclastogenesis required > or = 250 U per well and further increased as mMCSF rose higher. Our results demonstrate that osteoclast formation in the local bone environment is dominated by OPGL. Increasing mMCSF above basal levels does not further enhance osteoclast formation from BMMs, indicating that mMCSF does not play a dominant regulatory role in the bone marrow.

Internalization and half-life of membrane-bound macrophage colony-stimulating factor

The binding of recombinant human macrophage colony-stimulating factor (M-CSF) soluble receptor (rh-M-CSF-sR) to membrane-bound macrophage colony stimulating factor (m-M-CSF) and the internalization and recycling of rh-M-CSF-sR/m-M-CSF complexes mediated by m-M-CSF were studied with a model of J6-1 cell line. The results indicated that m-M-CSF bound rh-M-CSF-sR with high affinity (kd = 1.78×1012 mol/L) and mediated a temperature- and energydependent internalization of rh-M-CSF-sR, and that internalized rh-M-CSF-sR could return to the cell surface in an m-M-CSF-bound state, suggesting that m-M-CSF may have a capability to mediate the internalization and recycling of rh-M-CSF-sR/m-M-CSF complexes. In addition, the half-lives of cell-associated M-CSF and its receptor of stimulated normal human cord blood mononuclear cells (CBMCs) and 4 leukemic cell lines were measured by indirect immunofluorescence and flow cytometry. The results showed that the half-lives of the various kinds of M-CSF isoforms and M-CSF-R in 4 leukemic cells, except those of membrane-bound M-CSF receptor (M-CSF-mR) of K562 and HL-60 cells, were longer than those of corresponding M-CSF isoform and its receptor in stimulated normal human CBMCs, suggesting that there is a slow degradation rate of cell-associated M-CSF and its receptor in leukemic cells.

Signal transduction from membrane-bound macrophage colony-stimulating factor

Macrophage colony-stimulating factor (M-CSF) exists in different forms, which bind the same membrane receptor (M-CSFR), due to alternative splicing and post-translational modifications. Membrane bound M-CSF (mM-CSF) was highly expressed in some malignancies including J6-1, J6-2 (leukemia), SMMC7721, BEL7402 (hepatoma) cell lines and specimens from patients with leukemia, Hodgkin's Disease (HD), hepatoma, etc. (Zheng GG et al. Hematologica 1999, 84:951). Furthermore, we demonstrated that mM-CSF and its receptor played adhesion molecule-like roles in J6-1 cells and the activation of mM-CSF by recombinant soluble receptor (M-CSFsR) resulted in the decrease of cytoplasmic pH, mobilization of cytosolic calcium, tyrosine-phosphorylation on cytoplasmic protiens, etc. (Zheng GG et al. Leukemia Res 2000, in press). In the present study, we used cytochalasin B (CB) to study whether the integrity of cytoskeleton is necessary for mM-CSF signaling. The results showed that one hour's treatment of J6-1 cells by CB before exposed to M-CSFsR blocked the tyrosine-phosphorylation on cytoplasmic proteins with MW of 45 and 55-90kD, while decreased the sensitivity of cytoplasmic pH changes, but showed no obvious influence on cytosolic calcium mobilization. The initial activation process of most membrane proteins by their ligands involves dimerization or cluster of the membrane protein and cross-linking of the membrane protein by specific antibody can mimic the effects of their natural ligands, while dimerization or cluster is not indispensable for the signaling of some membrane proteins. We used a monoclonal antibody (McAb) B5, which was developed in our lab belonging to IgM subtype and recognizes N-terminal part of mM-CSF, to cross-link mM-CSF on J6-1 cell surface before underwent phospho-tyrosine Western blot analysis. While F(ab')2 fragment of B5 caused no obvious tyrosine phosphorylation when compared with BSA control, 10 minutes' treatment by B5 dose-dependently caused tyrosine phosphorylation on cytoplasmic proteins with MW of approximately 45 and 50-90kD, which mimicked the effects of M-CSFsR. The above results suggested that the initiation of mM-CSF signaling need dimerization/cluster of them on cell surface and cytoskeleton take part in some, but not all, of the downstream signal events.

Mechanical strain inhibits expression of osteoclast differentiation factor by murine stromal cells.

Normal dynamic loading prevents bone resorption; however, the means whereby biophysical factors reduce osteoclast activity are not understood. We show here that mechanical strain (2% at 10 cycles per minute) applied to murine marrow cultures reduced 1, 25(OH)(2)D(3)-stimulated osteoclast formation by 50%. This was preceded by decreased expression of osteoclast differentiation factor (ODF/TRANCE). RT-PCR for ODF/TRANCE revealed that ODF/TRANCE mRNA in strained cultures was 59 +/- 3% of that seen in control cultures. No significant effects on total cell count, thymidine uptake, or alkaline phosphatase activity were induced by strain. To isolate the cell targeted by strain, primary stromal cells were cultured from marrow. Mechanical strain also reduced mRNA for ODF/TRANCE to 60% that of control in these cells. In contrast, mRNA for membrane-bound macrophage colony-stimulating factor was not significantly affected. Soluble ODF ( approximately 2 ng/ml) was able to reverse the effect of strain, returning osteoclast numbers to control. Because osteoclast formation is dependent upon ODF/TRANCE expression, strain-induced reductions in this factor may contribute to the accompanying reduction in osteoclastogenesis.

Membrane-bound macrophage colony-stimulating factor and its receptor play adhesion molecule-like roles in leukemic cells

Membrane-bound macrophage colony-stimulating factor (m-M-CSF) is the membrane form M-CSF by alternative splicing. J6-1 leukemic cell line spontaneously forms cell clusters, whose growth depends on the auto-juxtacrine mediated by m-M-CSF and its receptor (M-CSFR). In this study, M-CSFR isolated from J6-1 cells and recombinant human M-CSF soluble receptor (rh-M-CSFsR) were used to study their effects on J6-1 cells. Both receptors inhibited cell proliferation. Use of M-CSFR monoclonal antibodies, M-CSFR or rh-M-CSFsR to block either M-CSFR or m-M-CSF on cell surface inhibited the cluster forming process, while both receptors stimulated cells adhering to culture plate. Furthermore, M-CSFR and/or rh-M-CSFsR caused multiple cellular changes including cytoplasmic pH, multinuclear cell ratio, antigen expression and cell diameter. A [Ca 2+] rise was induced within 90 s by both receptors. Western blot experiments showed that rh-M-CSFsR caused tyrosine phosphorylation on multiple cytoplasmic proteins of 45 kDa and 55–90 kDa, which could be blocked by H7. These observations suggested that m-M-CSF and M-CSFR mediate J6-1 cell intercellular adhesion with bi-directional signal transduction, and Ca 2+, protein tyrosine kinases, PKC and/or other H7 sensitive kinase(s) involve in the counter-directional signal transduction.

Receptor role of membrane bound macrophage colony-stimulating factor

According to the definition of cytokine, the direction of signaling should be from cytokine to receptor. The counter receptor was presented. Membrane bound macrophage colony-stimulating factor (m-M-CSF) and its receptor (M-CSF-R) were shown in human leukemic cell line J6-1 as autojuxtacrine mechanism. Soluble M-CSF receptor (sM-CSF-R), which was isolated from J6-1 cells membrane, was added into J6-1 cell culture. It was observed inhibition of J6-1 cell proliferation, decreasing of mitosis index and ratio of multinuclear cells, enlargement of cell diameter and volume, down regulation of numerous surface antigens. Dramatic change of intracellular pH was shown between several min to 20 min after treatment of sM-CSF-R. It suggested that some information was transmitted via m-M-CSF from sM-CSF-R. This counter signaling was not influenced by saccharification of m-MCSF.

Inhibition of bone resorption by 17β‐estradiol in human bone marrow cultures

Estrogen deficiency puts individuals at risk of developing osteoporosis because it causes increased bone resorption. However, the mechanism by which this occurs is not known. We have shown, using a recently described two-phase human bone marrow culture system, that estradiol (17β-E2) added to phase I results in inhibition of bone resorption by reducing the number of osteoclasts (identified as vitronectin receptor and/or calcitonin receptor-positive cells) formed in the cultures. The addition of 17β-E2 in phase II was without effect, indicating that it does not interfere with the bone resorptive process. 17β-E2 down-regulated mRNA expression and protein synthesis of the membrane form of macrophage colony-stimulating factor (M-CSF). 17β-E2 did not the alter the expression of the 4.0 kb M-CSF transcript. However, it increased protein synthesis of the proteoglycan form of M-CSF, but not the 85 kDa soluble form in the same cultures. Finally, addition of M-CSF to the cultures reversed the 17β-E2-induced inhibitory effect. These observations suggest that regulation of the synthesis of membrane-bound M-CSF plays a role in 17β-E2-induced inhibition of bone resorption. J. Cell. Physiol. 175:99–108, 1998. © 1998 Wiley-Liss, Inc.

Inhibition of bone resorption by 17beta-estradiol in human bone marrow cultures.

Estrogen deficiency puts individuals at risk of developing osteoporosis because it causes increased bone resorption. However, the mechanism by which this occurs is not known. We have shown, using a recently described two-phase human bone marrow culture system, that estradiol (17beta-E2) added to phase I results in inhibition of bone resorption by reducing the number of osteoclasts (identified as vitronectin receptor and/or calcitonin receptor-positive cells) formed in the cultures. The addition of 17beta-E2 in phase II was without effect, indicating that it does not interfere with the bone resorptive process. 17Beta-E2 down-regulated mRNA expression and protein synthesis of the membrane form of macrophage colony-stimulating factor (M-CSF). 17Beta-E2 did not the alter the expression of the 4.0 kb M-CSF transcript. However, it increased protein synthesis of the proteoglycan form of M-CSF, but not the 85 kDa soluble form in the same cultures. Finally, addition of M-CSF to the cultures reversed the 17beta-E2-induced inhibitory effect. These observations suggest that regulation of the synthesis of membrane-bound M-CSF plays a role in 17beta-E2-induced inhibition of bone resorption.

Dexamethasone Promotes Expression of Membrane-Bound Macrophage Colony-Stimulating Factor in Murine Osteoblast-Like Cells1

The mechanisms by which glucocorticosteroids promote osteoclastogenesis in vitro are uncertain. As macrophage colony-stimulating factor (MCSF) is critical for osteoclastogenesis, we hypothesized that glucocorticosteroids might regulate membrane-bound MCSF (mMCSF) and soluble MCSF (sMCSF) production by stromal cells or osteoblasts. ST2 cells or murine calvarial osteoblasts (MOBs) were treated with dexamethasone (Dex; 100 nM) and/or 1,25-dihydroxyvitamin D [1,25(OH)2D; 10 nM] for 3 days. Control values for mMCSF and sMCSF as units per 100,000 cells were 9 +/- 1.4 and 511 +/- 56 in ST2 cells and 5.9 +/- 0.8 and 379 +/- 47 in MOB cells, respectively. Dex increased mMCSF to 156 +/- 16% and 143 +/- 26% compared with the control value in ST2 and MOB cells, respectively, whereas 1,25-(OH)2D caused increases of 195 +/- 16% and 164 +/- 21%. In the presence of both Dex and 1,25-(OH)2D, mMCSF increased to 209 +/- 24% and 216 +/- 26% in the two cell types, respectively. 1,25-(OH)2D caused modest increases in sMCSF, as expected, in both cell types (153 +/- 6% and 122 +/- 4%). Dex inhibited 1,25-(OH)2D-stimulated sMCSF (115 +/- 7% of control) in ST2 cells. Analysis of mMCSF transcript levels by semiquantitative RT-PCR revealed Dex-stimulated increases of 170 +/- 11% in ST2 cells and 126 +/- 16% in MOB cells compared with the control level. The increased expression of the transcript for sMCSF in the presence of Dex and 1,25-(OH)2D, measured by both RT-PCR and Northern analysis (219 +/- 53% and 242%, respectively), despite inhibition of sMCSF protein, indicated that the inhibitory effect of Dex in ST2 cells was posttranscriptional. Half-life studies showed that Dex prolonged MCSF messenger RNA from 2.8 to 7.5 h. These results suggest that Dex influences osteoclastogenesis by increasing the expression of mMCSF by accessory cells in culture.

Dexamethasone Promotes Expression of Membrane-Bound Macrophage Colony-Stimulating Factor in Murine Osteoblast-Like Cells

The mechanisms by which glucocorticosteroids promote osteoclastogenesis in vitro are uncertain. As macrophage colony-stimulating factor (MCSF) is critical for osteoclastogenesis, we hypothesized that glucocorticosteroids might regulate membrane-bound MCSF (mMCSF) and soluble MCSF (sMCSF) production by stromal cells or osteoblasts. ST2 cells or murine calvarial osteoblasts (MOBs) were treated with dexamethasone (Dex; 100 nm) and/or 1,25-dihydroxyvitamin D[ 1,25(OH)2D; 10 nm] for 3 days. Control values for mMCSF and sMCSF as units per 100,000 cells were 9 ± 1.4 and 511 ± 56 in ST2 cells and 5.9 ± 0.8 and 379 ± 47 in MOB cells, respectively. Dex increased mMCSF to 156 ± 16% and 143 ± 26% compared with the control value in ST2 and MOB cells, respectively, whereas 1,25-(OH)2D caused increases of 195 ± 16% and 164 ± 21%. In the presence of both Dex and 1,25-(OH)2D, mMCSF increased to 209 ± 24% and 216 ± 26% in the two cell types, respectively. 1,25-(OH)2D caused modest increases in sMCSF, as expected, in both cell types (153 ± 6% and 122 ± 4%). Dex inhibited 1,25-(OH)2D-stimulated sMCSF (115 ± 7% of control) in ST2 cells. Analysis of mMCSF transcript levels by semiquantitative RT-PCR revealed Dex-stimulated increases of 170 ± 11% in ST2 cells and 126 ± 16% in MOB cells compared with the control level. The increased expression of the transcript for sMCSF in the presence of Dex and 1,25-(OH)2D, measured by both RT-PCR and Northern analysis (219 ± 53% and 242%, respectively), despite inhibition of sMCSF protein, indicated that the inhibitory effect of Dex in ST2 cells was posttranscriptional. Half-life studies showed that Dex prolonged MCSF messenger RNA from 2.8 to 7.5 h. These results suggest that Dex influences osteoclastogenesis by increasing the expression of mMCSF by accessory cells in culture.

Human macrophage colony-stimulating factor is expressed at and shed from the cell surface

Surface membrane-associated growth factors are being recognized as important for developmental processes, including cell assembly, differentiation, and growth. To investigate the role of membrane-bound macrophage colony-stimulating factor (M-CSF) in myelopoiesis, and whether this factor is released from the cell surface in association with shed membrane-derived vesicles, COS-1 cells were transfected with cDNAs for M-CSF-tau (containing the transmembrane domain) or a soluble mutant form of the molecule lacking the transmembrane domain ([s]M-CSF- alpha). COS-1 cells transfected with either cDNA released activity into the spent culture medium. Conditioned medium was separated by centrifugation into supernatants and pellets were found to contain plasma membrane-derived vesicles by transmission electron microscopy. When medium fractions were assayed in marrow cultures, activity was localized to shed plasma membrane-derived vesicles in medium conditioned by cells transfected with cDNA for M-CSF-tau and in the vesicle-free supernatants of medium conditioned by cells transfected with cDNA for [s]M-CSF-alpha. In addition, nuclear, mitochondrial, and plasma membrane subfractions of stably transfected cells were prepared and assayed for activity. Concentration-dependent stimulation of macrophage colony formation was observed with purified plasma membranes (but not nuclear or cytosolic proteins) from cells transfected with cDNA for M-CSF-tau. By contrast, membranes from untransfected cells and cells transfected with cDNA for [s]M-CSF-alpha or control DNA expressed no activity. Together, the data indicate that human M-CSF is expressed at the cell surface and exfoliated in association with surface membrane- derived vesicles.

Human Macrophage Colony-Stimulating Factor Is Expressed at and Shed From the Cell Surface

Surface membrane-associated growth factors are being recognized as important for developmental processes, including cell assembly, differentiation, and growth. To investigate the role of membrane-bound macrophage colony-stimulating factor (M-CSF) in myelopoiesis, and whether this factor is released from the cell surface in association with shed membrane-derived vesicles, COS-1 cells were transfected with cDNAs for M-CSF-tau (containing the transmembrane domain) or a soluble mutant form of the molecule lacking the transmembrane domain ([s]M-CSF-alpha). COS-1 cells transfected with either cDNA released activity into the spent culture medium. Conditioned medium was separated by centrifugation into supernatants and pellets were found to contain plasma membrane-derived vesicles by transmission electron microscopy. When medium fractions were assayed in marrow cultures, activity was localized to shed plasma membrane-derived vesicles in medium conditioned by cells transfected with cDNA for M-CSF-tau and in the vesicle-free supernatants of medium conditioned by cells transfected with cDNA for [s]M-CSF-alpha. In addition, nuclear, mitochondrial, and plasma membrane subfractions of stably transfected cells were prepared and assayed for activity. Concentration-dependent stimulation of macrophage colony formation was observed with purified plasma membranes (but not nuclear or cytosolic proteins) from cells transfected with cDNA for M-CSF-tau. By contrast, membranes from untransfected cells and cells transfected with cDNA for [s]M-CSF-alpha or control DNA expressed no activity. Together, the data indicate that human M-CSF is expressed at the cell surface and exfoliated in association with surface membrane-derived vesicles.

Binding of membrane-anchored macrophage colony-stimulating factor (M- CSF) to its receptor mediates specific adhesion between stromal cells and M-CSF receptor-bearing hematopoietic cells

To explore the biologic significance of the membrane-anchored form of macrophage colony-stimulating factor (M-CSF), we examined whether interaction between membrane-bound M-CSF and its receptor mediates intercellular adhesion as well as cell proliferation and differentiation. Human M-CSF receptors were expressed on a murine interleukin-2 (IL-3)-dependent cell line, FDC-P2, by DNA transfection with the c-fms gene (FDC-P2-MCSFR). A human bone marrow-derived stromal cell line, KM102, was used in the cell adhesion assay. The expression of membrane-bound M-CSF on KM102 cells was demonstrated by flow cytometry and immunoblot analysis. After the incubation of parent and transformed FDC-P2 cells on confluent KM102 cells, nonadherent cells were removed and the cells attached to KM102 cells were examined microscopically. Almost all parent FDC-P2 cells were nonadherent, whereas a significant number of FDC-P2-MCSFR cells bound to KM102 cells. The addition of anti-M-CSF or anti-M-CSF receptor neutralizing antibodies dose-dependently inhibited the binding of [3H]-thymidine- labeled FDC-P2-MCSFR cells to KM102 cells. An excess amount of M-CSF also inhibited the binding. On the other hand, the addition of antibodies against some representative adhesion molecules (vitronectin receptor, Pgp-1/CD44, and VLA-4) did not inhibit the adhesion between FDC-P2-MCSFR cells and KM102 cells. These results support the idea that membrane-anchored M-CSF and its receptor may function as mediators of cell-cell adhesion.