Activity Of Macrophage Colony-stimulating Factor Research Articles

Macrophage colony stimulating factor (MCSF) of Japanese flounder (Paralichthys olivaceus): Immunoregulatory property, anti-infectious function, and interaction with MCSF receptor

Macrophage colony-stimulating factor (MCSF) is an essential growth factor to control the proliferation, differentiation and survival of the macrophage lineage in vertebrates. Sequences of MCSF have been identified in multiple teleost species, however, the functional investigations of MCSF were documented in only a few species. In this study, we examined the biological activity and the immunomodulatory property of a MCSF homologue, PoMCSF, from Japanese flounder (Paralichthys olivaceus). Structural analysis showed that PoMCSF possesses conserved structural characteristics of MCSF proteins, including a signal peptide, a CSF-1 domain, and a transmembrane region closed to the C-terminal. Under normal physiological condition, PoMCSF expression distributes in all the examined tissues, the highest three tissues are blood, muscle, and head kidney. When infected by extracellular and intracellular bacterial pathogens and viral pathogen, the PoMCSF expression patterns vary with different types of microbial pathogens infection and different immune tissues. In vitro experiment showed recombinant PoMCSF promoted the activity of macrophage. In vivo experiment indicated that PoMCSF overexpression boosted the defensive ability of flounder against Edwardsiella piscicida, a severe fish pathogen that infects multiple species of economically important fish, and regulated the expression of multiple immune-related genes. To explore the relationship between PoMCSF and its receptor PoMCSFR, anti-PoMCSFR antibody was prepared and PoMCSFR knockdown was conducted. The neutralization assay showed that when PoMCSFR was neutralized by its antibody, the role of PoMCSF on host defense against E. piscicida was weakened. Knockdown of PoMCSFR impaired the phagocytic capacity of macrophages. Collectively, these findings suggest that PoMCSF plays a crucial role in the immune defense system of Japanese flounder and the effect of PoMCSF is dependent on PoMCSFR. This study provides new insights into the biological activity of MCSF and the relationship between MCSF and MCSFR in teleost.

Role of macrophage colony-stimulating factor in the development of neointimal thickening following arterial injury.

Evidence suggests that macrophage colony-stimulating factor (M-CSF) participates critically in atherosclerosis; little is known about the role of M-CSF in the development of neointimal hyperplasia following mechanical vascular injury. We examined the expression of M-CSF and its receptor, c-fms, in rodent and rabbit models of arterial injury. Injured rat carotid arteries expressed 3- to 10-fold higher levels of M-CSF and c-fms mRNA and protein following balloon injury as compared to uninjured arteries. In the rabbit, M-CSF protein expression was greatest in neointimal smooth muscle cells (SMCs) postinjury, with some expression in medial SMCs. M-CSF-positive SMCs exhibited markers of proliferation. At 30days postinjury, neointimal SMCs in the adjacent healed area near the border between injured and uninjured zone lost both proliferative activity and overexpression of M-CSF. The presence of induced M-CSF and c-fms expression correlated with the initiation of SMCs proliferation. M-CSF stimulated incorporation of [(3)H] thymidine in human aortic smooth muscle cells in a concentration-dependent manner. Serum-free conditioned medium from aortic SMCs also promoted DNA synthesis, and this effect was blocked by M-CSF specific antibody. To test further the role of M-CSF in vivo, we induced arterial injury by placing a periadventitial collar around the carotid arteries in compound mutant mice lacking apolipoprotein apoE (apoE(-/-)) and M-CSF. Loss of M-CSF abolished the neointimal hyperplastic response to arterial injury in apoE(-/-) mice. Local delivery of M-CSF to the injured artery restored neointimal proliferation, suggesting a critical role of M-CSF for the development of neointimal thickening following arterial injury.

IFN-γ–mediated inhibition of MAPK phosphatase expression results in prolonged MAPK activity in response to M-CSF and inhibition of proliferation

AbstractMacrophages have the capacity to proliferate in response to specific growth factors, such as macrophage-colony stimulating factor (M-CSF). In the presence of several cytokines and activating factors, macrophages undergo growth arrest, become activated, and participate in the development of an immune response. We have previously observed that activation of extracellularly regulated kinase 1/2 (ERK-1/2) is required for macrophage proliferation in response to growth factors. A short and early pattern of ERK activity correlated with the proliferative response. In contrast, slightly prolonged patterns of activity of these kinases were induced by signals that lead to macrophage activation and growth arrest. IFN-γ is the main endogenous Th1-type macrophage activator. Here we report that stimulation with IFN-γ prolongs the pattern of ERK activity induced by M-CSF in macrophages. These effects correlate with IFN-γ–mediated inhibition of the expression of several members of the MAPK phosphatase family, namely MKP-1, -2, and -4. Moreover, inhibition of MKP-1 expression using siRNA technology or synthetic inhibitors also led to elongated ERK activity and significant blockage of M-CSF–dependent proliferation. These data suggest that subtle changes in the time course of activity of members of the MAPK family contribute to the antiproliferative effects of IFN-γ in macrophages.

Interaction between Hck and HIV-1 Nef negatively regulates cell surface expression of M-CSF receptor

Nef is a multifunctional pathogenetic protein of HIV-1, the interaction of which with Hck, a Src tyrosine kinase highly expressed in macrophages, has been shown to be responsible for the development of AIDS. However, how the Nef-Hck interaction leads to the functional aberration of macrophages is poorly understood. We recently showed that Nef markedly inhibited the activity of macrophage colony-stimulating factor (M-CSF), a primary cytokine for macrophages. Here, we show that the inhibitory effect of Nef is due to the Hck-dependent down-regulation of the cell surface expression of M-CSF receptor Fms. In the presence of Hck, Nef induced the accumulation of an immature under-N-glycosylated Fms at the Golgi, thereby down-regulating Fms. The activation of Hck by the direct interaction with Nef was indispensable for the down-regulation. Unexpectedly, the accumulation of the active Hck at the Golgi where Nef prelocalized was likely to be another critical determinant of the function of Nef, because the expression of the constitutive-active forms of Hck alone did not fully down-regulate Fms. These results suggest that Nef perturbs the intracellular maturation and the trafficking of nascent Fms, through a unique mechanism that required both the activation of Hck and the aberrant spatial regulation of the active Hck.

Effects of Vitaxin®, a novel therapeutic in trial for metastatic bone tumors, on osteoclast functions in vitro

The integrin alphavbeta3 mediates cell-matrix interactions. Vitaxin(R), a humanized monoclonal antibody that blocks human and rabbit alphavbeta3 integrins, is in clinical trials for metastatic melanoma and prostate cancer. alphavbeta3 is the predominant integrin on osteoclasts, the cells responsible for bone resorption in health and disease. Here, we report the first investigation of Vitaxin's effects on osteoclast activity. Vitaxin (100-300 ng/ml) decreased total resorption by 50%, but did not alter resorptive activity per osteoclast. Vitaxin (300 ng/ml) decreased osteoclast numbers on plastic by 35% after 48 h. Similarly, attachment after 2 h was reduced by 30% when osteoclasts were incubated with Vitaxin (300 ng/ml) for 25 min prior to plating; however, the rate of fusion of osteoclast precursors in Vitaxin-treated and control groups was equal. Using time-lapse microscopy, we evaluated the effect of Vitaxin on osteoclast morphology and found a significant reduction in osteoclast planar area only when cells were pretreated with macrophage colony stimulating factor (M-CSF). Extracellular Ca(2+) and M-CSF have opposite effects on alphavbeta3 conformation. Elevation of extracellular Ca(2+) eliminated the inhibitory effect of Vitaxin on osteoclast attachment. In contrast, the effect of Vitaxin was enhanced in cells pretreated with M-CSF. This action of M-CSF was suppressed by the phosphatidylinositol 3-kinase (PI3-kinase) inhibitor wortmannin, suggesting that M-CSF increases Vitaxin's inhibitory effect by inside-out activation of alphavbeta3. In conclusion, Vitaxin decreases resorption by impairing osteoclast attachment, without affecting osteoclast formation and multinucleation. Our data also show that Vitaxin's inhibitory effects on osteoclasts can be modulated by factors known to alter the conformation of alphavbeta3.

Macrophage-colony stimulating factor enhances MHC-restricted presentation of exogenous antigen in dendritic cells

Previous studies have shown that dendritic cells (DCs) can phagocytize, process and present a microencapsulated form of ovalbumin (OVA) in the context of class I MHC as well as class II MHC. In the present study, we examined the effects of recombinant human macrophage-colony stimulating factor (M-CSF) on the MHC-restricted presentation of microencapsulated OVA by DCs. Two types of DCs were generated from mouse bone marrow (BM) cells, one type with granulocyte/macrophage-colony stimulating factor (GM-CSF) alone, the other type with GM-CSF and interleukin (IL)-4. Pretreatment with M-CSF significantly enhanced both class I MHC and class II MHC-restricted presentation of exogenous OVA by both types of DCs. The enhancing activity of M-CSF on antigen presentation was more potent in DCs generated with GM-CSF alone compared to DCs generated with both GM-CSF and IL-4. Pretreatment of the DCs with M-CSF did not increase phagocytic activity or total level of expression of class I MHC (H-2K b) molecules, but increased expression of OVA peptide-H-2K b complexes upon phagocytosis of microencapsulated OVA. These results demonstrate that M-CSF increases intracellular processing events of phagocytized antigen in DCs.

Effect of cytokines on the proliferation/differentiation of stroma-initiating cells.

A culture system that identifies the precursor of murine bone marrow fibroblastic stromal cells (stroma-initiating cells, SIC) has been developed. In this system, mature fibroblasts are depleted by adherence to plastic dishes and the nonadherent cells are seeded at a low density, which results in the formation of colonies composed of fibroblastic cells. Macrophage colony-stimulating factor (M-CSF) has been shown to accelerate the colony formation in the system. In this study, we examined the stroma-inducing activity of a number of cytokines. Neither granulocyte-CSF, stem cell factor, interleukin (IL)-1, IL-6, transforming growth factor, epidermal growth factor, insulin-like growth factor, platelet-derived growth factor, nor fibroblast growth factor showed the activity. Similarly, tumor necrosis factor (TNF) did not show any stroma-inducing activity, but the factor inhibited the stromal colony formation induced by M-CSF. In this study, we found that granulocyte/macrophage-CSF (GM-CSF) and IL-3, as well as M-CSF had the stroma-inducing activity. Neither an additive nor synergistic effect was observed when the three factors were assayed in various combinations. The stroma-inducing activity of M-CSF, GM-CSF and IL-3 was observed even if lineage-negative bone marrow cells were used as target cells, suggesting that mature hematopoietic cells such as macrophages and granulocytes were not involved in the induction of stromal colony formation by these factors. Our results raise the possibility that GM-CSF and IL-3 as well as M-CSF stimulate the proliferation or differentiation of the precursor of bone marrow fibroblastic stromal cells.

M-CSF and GM-CSF promote alveolar macrophage differentiation into multinucleated giant cells with distinct phenotypes.

Multinucleated giant cells (MGC) are a hallmark of granulomatous reactions but the mechanisms that regulate their formation are unknown. To address this issue, we cultured resident alveolar macrophages (AM) from rat lung and examined the effects of defined cytokines on AM differentiation and MGC formation. The presence of MGC was found after 3 days in culture with maximal numbers obtained at 7 days and thereafter (up to 21 days). Macrophage colony-stimulating factor (M-CSF) and granulocyte-macrophage colony-stimulating factor (GM-CSF) (25-75 U/mL) stimulated the formation of MGC (up to 4-fold), whereas interleukin (IL) -3, IL-10, and interferon-gamma (IFN-gamma) had no stimulatory effect. Interestingly, MGC with distinct phenotypes were observed in AM cultures: (1) spherical MGC with 3-16 nuclei, dense cytoplasm, and lower expression of beta3 integrin (Type 1) and (2) irregular MGC with 3-30 nuclei, thin and vacuolated cytoplasm, and higher expression of beta3 integrin (Type 2). Furthermore, the actions of M-CSF and GM-CSF on AM were found to be different. GM-CSF promoted, in AM cultures, the appearance of an elongated fibroblastoid phenotype and stimulated mostly the formation of Type 2 MGC. In contrast, M-CSF did not cause significant change in the general morphology of regular AM but stimulated the appearance of both Type 1 and Type 2 MGC. Reverse transcriptase-polymerase chain reaction analysis demonstrated that, under these conditions, M-CSF induced GM-CSF gene expression in AM. In addition, neutralizing antibodies against M-CSF selectively decreased the formation of Type 1 MGC, whereas neutralizing anti-GM-CSF inhibited Type 2 formation. These data suggest that M-CSF promotes AM differentiation into Type 1 MGC, whereas GM-CSF stimulates the formation of Type 2 and that M-CSF and GM-CSF may selectively regulate in an autocrine fashion AM differentiation into distinct MGC.

Effect of recombinant human macrophage-colony stimulating factor on marrow, splenic, and peripheral hematopoietic progenitor cells in mice.

The effects of human macrophage colony-stimulating factor (M-CSF) on marrow, splenic, and peripheral progenitor cells (CFU-M, CFU-GM, and CFU-G) were investigated in mice administered recombinant human M-CSF (8-4,000 micrograms/kg). Single injection of 4,000 micrograms/kg of M-CSF resulted in a decrease in the number of marrow progenitor cells (CFU-M, CFU-GM, and CFU-G) on day 2 followed by a gradual increase, returning to the original level on day 4 or 5. In contrast, each type of splenic progenitors tested for started to increase markedly on day 2, reaching a level 4- to 15-fold higher than that of the basal value on day 3 or 4. Peripheral CFU-M, CFU-GM, and CFU-G also increased on day 2. In addition, administration of 800 micrograms/kg of M-CSF in mice caused a decrease in marrow CFU-G, as well as an increase in splenic CFU-G. The present results indicate that treatments of mice with pharmacological concentrations of human M-CSF affect the number of progenitor cells not only of monocyte/macrophage lineage but also of granulocyte lineage. Also, the coincidence between decrease of marrow progenitor cells and increase of splenic and peripheral progenitor cells suggests that the progenitor cells are released from bone marrow to peripheral blood and reseeded to the spleen by the action of M-CSF.

Low-density lipoprotein stimulates the expression of macrophage colony-stimulating factor in glomerular mesangial cells

Disordered lipoprotein metabolism and the enhanced influx and accumulation of circulating mononuclear leukocytes into vascular tissue are common pathobiological phenomena associated with both atherosclerosis and glomerulosclerosis. Since atherogenic lipoproteins (such as low density lipoprotein, LDL) have been implicated in monocyte migration and proliferation into the glomerular mesangium, we examined the effect of LDL on mesangial cell expression of macrophage colony-stimulating factor (M-CSF), a cytoregulatory peptide associated with monocyte chemoattraction, differentiation and proliferation. Mesangial cell M-CSF gene expression, protein synthesis and secretion, and its biological activity to induce progenitor colony formation and monocyte proliferation were studied in murine mesangial cells. Incubation of either primary cultures or SV-40 transformed murine mesangial cells with LDL (0 to 200 micrograms/ml) induced M-CSF steady-state mRNA expression, in a dose-dependent manner (52 to 183% of control) when Northern blots were analyzed quantitatively by densitometric scanning. Similarly, Western blot analysis showed that LDL-activated SV-40 transformed mesangial cells increased M-CSF protein synthesis and secretion in a dose-dependent manner. The conditioned media obtained by incubating mesangial cells with LDL induced bone marrow progenitor colony formation that could be inhibited by specific neutralizing antibodies against murine M-CSF. Finally, the biological activity of M-CSF secreted by LDL-activated mesangial cells was further confirmed by its enhanced ability to induce monocyte proliferation. These data indicate that LDL, by activating mesangial cells to induce M-CSF and possibly other monocyte chemoattractants, may regulate the migration and proliferation of cells of mononuclear leukocytic origin into the mesangium supporting a pathobiological role for LDL in glomerular injury.

Phase II Trial of Recombinant Human Macrophage Colony-Stimulating Factor in Metastatic Soft Tissue Sarcoma

This Phase II study was undertaken to access the activity of recombinant human macrophage colony stimulating factor (M-CSF) in metastatic soft tissue sarcoma based on the observation of a partial response in a patient with leiomyosarcoma during an earlier Phase I trial. Fifteen patients with metastatic soft tissue sarcoma (seven males and eight females) were entered on the trial between October 1990 and March 1991. Seven of these patients had leiomyosarcoma. One mg/M2 of M-CSF was administered by rapid intravenous infusion every 8 h on days 1-5 and 15-19. Treatment cycles were repeated at 35-day intervals. Patients were evaluated initially for response after the first cycle, and then following alternate cycles. One partial response was observed in a patient with metastatic small bowel leiomyosarcoma (response rate 7%, 95% confidence interval, 0-33%). Two additional patients had stable disease for 10-15 months on study. Four patients had clinically significant bleeding from tumor sites during M-CSF therapy. No evidence of toxicity directly attributable to M-CSF was observed in any patient. Mean monocyte counts increased in patients during the first 20 days of treatment (p = 0.013). At this dose and schedule, meaningful activity of M-CSF in previously treated patients with soft tissue sarcoma could not be demonstrated. However, the activity observed in patients with leiomyosarcoma in this trial and in the previous Phase I study are intriguing. Further studies of M-CSF in previously untreated patients with leiomyosarcoma may be warranted.

Cytokine-mediated protein kinase C activation is a signal for lineage determination in bipotential granulocyte macrophage colony-forming cells.

Granulocyte macrophage colony-forming cells (GM-CFC) have the potential to develop into either macrophages and/or neutrophils. With a highly enriched population of these cells we have found that although GM-CFC are equally responsive to macrophage colony stimulating factor (M-CSF) and stem cell factor (SCF) in terms of DNA synthesis, M-CSF stimulated the development of colonies containing macrophages in soft gel assays, while SCF promoted neutrophilic colony formation. When SCF and M-CSF were combined, mainly macrophage development was stimulated both in soft agar colony-forming assays and liquid cultures. An analysis of some potential signaling mechanisms associated with cytokine-mediated developmental decisions in GM-CFC revealed that M-CSF, but not SCF, was able to chronically stimulate phosphatidylcholine breakdown and diacylglycerol production, indicating that protein kinase C (PKC) may be involved in the action of M-CSF. Furthermore, M-CSF, but not SCF, can increase the levels of PKC alpha (PKC alpha) expression and stimulate the translocation of PKC alpha to the nucleus. When the PKC inhibitor, calphostin C, was added to GM-CFC cultured in M-CSF then predominantly neutrophils were produced, conversely PKC activators added with SCF stimulated macrophage development. The data indicate a role for PKC in M-CSF-stimulated macrophage development from GM-CFC.

Timing in administration of a heat-killed Lactobacillus casei preparation for radioprotection in mice.

A single subcutaneous injection of a preparation of heat-killed Lactobacillus casei (LC 9018), given before or after irradiation, significantly increased the survival rate of mice that had received 8.5-Gy 137Cs whole-body gamma-irradiation. A similar radioprotective effect was observed when LC 9018 was administered within the period from 2 days before irradiation to 9 h after irradiation, the pre-irradiation treatment being slightly better than the post-irradiation treatment. Increases in the weight of the spleen and in the number of endogenous spleen colonies on days 8 and 12 after irradiation suggested that the radioprotective effect was based on enhanced recovery of hematopoietic tissues. The activity of macrophage colony-stimulating factor (M-CSF) in serum was rapidly increased by the treatment and was maintained at the elevated level for 13 days. At the same time, an increased level of M-CSF mRNA was detected in the livers of the treated mice. However, LC 9018 failed to save the lives of mice when administered 3 days after irradiation, although it increased serum M-CSF as effectively as noted above. The small advantage of the pre-irradiation over the post-irradiation treatment was not explained by the increases of metallothionein in the hematopoietic tissues of the treated mice.

Macrophage Colony-Stimulating Factor (M-CSF) Induces Migration in Osteoclasts in Vitro

It has recently been shown that M-CSF is essential for osteoclast formation and survival. Paradoxically, the cytokine inhibits bone resorption by isolated osteoclasts. This implies that it may stimulate some function in osteoclasts not directly related to the bone resorptive process itself. We now report that M-CSF stimulates migration in isolated rat osteoclasts. This was achieved predominantly by a 3-8-fold increase in the proportion of osteoclasts demonstrating migration, combined with an increase in the area of substrate covered (43-96%) by each migrating cell. This action of M-CSF on migration is the inverse of its effect on resorption, wherein it has been previously found to reduce the proportion of osteoclasts that resorb bone and suggests that migration and resorption might represent alternative states of osteoclast behaviour. The results also suggest that osteoblast-derived M-CSF might play a role in the induction and regulation of localisation of these cells in bone.

Macrophage colony-stimulating factor is indispensable for both proliferation and differentiation of osteoclast progenitors.

The mechanism of action of macrophage colony-stimulating factor (M-CSF) in osteoclast development was examined in a co-culture system of mouse osteoblastic cells and spleen cells. In this co-culture, osteoclast-like multinucleated cells (MNCs) were formed within 6 d in response to 10 nM 1 alpha,25(OH)2D3 added only for the final 2 d of culture. Simultaneously adding hydroxyurea for the final 2 d completely inhibited proliferation of cultured cells without affecting 1 alpha,25(OH)2D3-stimulated MNC formation. Autoradiographic examination using [3H]-thymidine revealed that osteoclast progenitors primarily proliferated during the first 4 d, whereas their differentiation into MNCs occurred predominantly during the final 2 d of culture in response to 1 alpha,25(OH)2D3. When anti-M-CSF antibody or anti-M-CSF receptor antibody was added either for the first 4 d or for the final 2 d, the MNC formation was similarly inhibited. In co-cultures of normal spleen cells and osteoblastic cells obtained from op/op mice, which cannot produce functionally active M-CSF, the lack of M-CSF either for the first 4 d or for the final 2 d failed to form MNCs in response to 1 alpha,25(OH)2D3 added for the last 2 d. These results clearly indicate that M-CSF is indispensable for both proliferation of osteoclast progenitors and their differentiation into mature osteoclasts.

Detection of transcripts for the receptor for macrophage colony-stimulating factor, c-fms, in murine osteoclasts.

Macrophage colony-stimulating factor (M-CSF), whose action is restricted to the cell populations of the mononuclear phagocyte system, has recently been found to be required for osteoclastogenesis and bone resorption. To investigate the cells involved in the action of M-CSF in these processes, expression of c-fms mRNA, encoding the M-CSF receptor, was studied by in situ hybridization. Paws from murine embryos and newborn mice, tibiae from 2-day-old animals, as well as isolated osteoclasts, were hybridized with a c-fms-specific RNA probe. In bone, c-fms mRNA was detected only in cells at the late stages of osteoclastogenesis and in mature osteoclasts. The findings strengthen the relation between osteoclasts and the mononuclear phagocyte system. Furthermore, they suggest that M-CSF acts directly on osteoclast precursors and on mature osteoclasts during osteoclastogenesis.

Production of macrophage colony-stimulating factor by adult murine parenchymal liver cells (hepatocytes).

The activity of macrophage colony-stimulating factor (M-CSF) was found in the culture supernatant of mouse parenchymal liver cell fractions in a bone marrow colony-forming assay. The activity of an M-CSF-like substance purified by a four-step procedure was neutralized by goat anti-mouse M-CSF antiserum. M-CSF mRNA was detected in cellular RNA prepared from cultured parenchymal liver cell fractions by Northern blot analysis and also in cultured parenchymal liver cells by in situ hybridization. These results indicate that parenchymal liver cells have the capacity to produce M-CSF. We discuss the role of M-CSF in hematopoiesis, the immune response, and other biological phenomena.

Purification of human marrow progenitor cells and demonstration of the direct action of macrophage colony-stimulating factor on colony-forming unit-macrophage

To facilitate the investigation of the direct interaction between hematopoietic progenitors and colony-stimulating factors, we have developed a method to purify human marrow progenitor cells. Using density centrifugation, negative panning with concanavalin A coated plates, positive selection of CD34-positive cells with immunomagnetic microspheres, overnight adherence to a plastic dish, negative selection with a panel of monoclonal antibodies, and density centrifugation, human marrow progenitor cells were purified from 1.5% to 53.2%, a 42- fold purification, with a 4.8% yield. The purified cells consisted of 38% erythroid, 9% colony forming unit-granulocyte (CFU-G), 29% CFU- macrophage (CFU-M), 12% CFU-eosinophil/basophil (CFU-Eo/Ba), and 4% CFU- mix. The purified cells cultured in serum-free fibrin clots with recombinant human macrophage colony-stimulating factor (rM-CSF) for 14 days developed a pure population of CFU-M colonies. An appearance of CFU-M colonies was present after the addition of 1 U/mL of rM-CSF and the maximum stimulation was found at 100 U/mL. When the purified cells were cultured in serum-free medium with rM-CSF in a limiting dilution assay and the percentage of nonresponder wells for CFU-M colonies was plotted against cell concentration, serum-free cultures yielded a straight line through the origin, indicating that CFU-M development did not depend on accessory cells and that rM-CSF acted directly on the CFU- M.

Purification of Human Marrow Progenitor Cells and Demonstration of the Direct Action of Macrophage Colony-Stimulating Factor on Colony-Forming Unit-Macrophage

To facilitate the investigation of the direct interaction between hematopoietic progenitors and colony-stimulating factors, we have developed a method to purify human marrow progenitor cells. Using density centrifugation, negative panning with concanavalin A coated plates, positive selection of CD34-positive cells with immunomagnetic microspheres, overnight adherence to a plastic dish, negative selection with a panel of monoclonal antibodies, and density centrifugation, human marrow progenitor cells were purified from 1.5% to 53.2%, a 42-fold purification, with a 4.8% yield. The purified cells consisted of 38% erythroid, 9% colony forming unit-granulocyte (CFU-G), 29% CFU-macrophage (CFU-M), 12% CFU-eosinophil/basophil (CFU-Eo/Ba), and 4% CFU-mix. The purified cells cultured in serum-free fibrin clots with recombinant human macrophage colony-stimulating factor (rM-CSF) for 14 days developed a pure population of CFU-M colonies. An appearance of CFU-M colonies was present after the addition of 1 U/mL of rM-CSF and the maximum stimulation was found at 100 U/mL. When the purified cells were cultured in serum-free medium with rM-CSF in a limiting dilution assay and the percentage of nonresponder wells for CFU-M colonies was plotted against cell concentration, serum-free cultures yielded a straight line through the origin, indicating that CFU-M development did not depend on accessory cells and that rM-CSF acted directly on the CFU-M.