Cultured Rat Bone Marrow Research Articles

Arylsulfatase B activities and glycosaminoglycan levels in retrovirally transduced mucopolysaccharidosis type VI cells. Prospects for gene therapy.

Mucopolysacchariodosis type VI (MPS VI) is the lysosomal storage disorder caused by the deficient activity of arylsulfatase B (ASB; N-acetylgalactosamine 4-sulfatase) and the subsequent accumulation of the glycosaminoglycan (GAG), dermatan sulfate. In this study, a retroviral vector containing the full-length human ASB cDNA was constructed and used to transduce skin fibroblasts, chondrocytes, and bone marrow cells from human patients, cats, or rats with MPS VI. The ASB vector expressed high levels of enzymatic activity in each of the cell types tested and, in the case of cat and rat cells, enzymatic expression led to complete normalization of 35SO4 incorporation. In contrast, overexpression of ASB in human MPS VI skin fibroblasts did not lead to metabolic correction. High-level ASB expression was detected for up to eight weeks in transduced MPS VI cat and rat bone marrow cultures, and PCR analysis demonstrated retroviral-mediated gene transfer to approximately 30-50% of the CFU GM-derived colonies. Notably, overexpression of ASB in bone marrow cells led to release of the enzyme into the media and uptake by MPS VI cat and rat skin fibroblasts and/or chondrocytes via the mannose-6-phosphate receptor system, leading to metabolic correction. Thus, these studies provide important rationale for the development of gene therapy for this disorder and lay the frame-work for future in vivo studies in the animal model systems.

Voltage-dependent potentiation of low-voltage-activated Ca2+ channel currents in cultured rat bone marrow cells.

1. The whole-cell patch-clamp technique was used to study Ca2+ channel currents in stromal cells of 7-10 day dexamethasone-treated and control rat bone marrow cultures. In saline containing either 108 mM Ba2+ or a 2.5 mM Ca(2+)-1 mM Mg2+ mixture, most cells expressed both fast-inactivating, low-voltage-activated (LVA) and slow-inactivating, high-voltage-activated (HVA) currents. 2. Repeated application of 400 ms voltage steps to 60 mV above the holding potential (Vh, -90 mV in Ca(2+)-Mg2+ mixture and -60 mV in Ba2+) at a frequency > or = 0.1 Hz resulted in a potentiation of the LVA component of the 2nd and subsequent currents. 3. LVA current potentiation was examined using a two-pulse (prepulse-test pulse) method. Prepulses to Vh + 150 mV induced an 80-100% increase in the amplitude of the LVA component of Ca2+ channel currents in saline containing either Ba2+ or Ca(2+)-Mg2+. This effect was also seen in non-dexamethasone-treated cultures. 4. Potentiation was not modified by omission of ATP and GTP from the pipette saline, and was not inhibited by extracellular application of the broad spectrum kinase inhibitors H-7 or RK252-a. 5. Potentiation was dependent on the amplitude and duration of the prepulse. Using the standard protocol, the threshold for potentiation was approximately Vh + 45 mV and saturation occurred at Vh + 150-180 mV. Further increases in prepulse amplitude did not modify potentiation. With a prepulse to +10 mV (Ba2+ saline) potentiation was half-maximal with a prepulse duration of 250-300 ms duration and saturated at 750-1000 ms. 6. Peak potentiation occurred 1-2 s after the prepulse. The time for total decay of potentiation varied from 10 to 90 s. 7. Voltage dependency of prepulse-induced potentiation did not resemble that of inactivation induced by similar prepulses. 8. Current kinetics, I-V relationship and sensitivity to blockade by Ni2+ and diphenylhydantoin of prepulse-recruited current resembled those of control LVA current. 9. The amplitude of prepulse-recruited current was positively correlated with control LVA current amplitude. 10. LVA currents supported regenerative potentials under current clamp. Repeated activation reduced spike latency. 11. It is suggested that current potentiation may be recruited physiologically, possibly in association with activation of stretch-sensitive channels, causing enhanced activation of HVA Ca2+ currents.

Interleukin‐10 selectively inhibits osteoclastogenesis by inhibiting differentiation of osteoclast progenitors into preosteoclast‐like cells in rat bone marrow culture system

Recombinant human interleukin-10 (hIL-10) inhibited the formation of osteoclast-like multinucleated cells in rat whole bone marrow cultures. The effect of hIL-10 on the process of osteoclast formation was further examined, since the process of osteoclast formation includes the proliferation and the differentiation of osteoclast progenitors into mononuclear preosteoclasts and the fusion of preosteoclasts into multinucleated osteoclasts. In the nonadherent bone marrow cell culture system, which was free of stromal cells and formed preosteoclast-like cells, hIL-10 significantly inhibited the formation of preosteoclast-like cells even at a very low concentration (0.5 U/ml). The strong inhibition appeared even after treatment with hIL-10 for only the first 24 h of the culture. However, hIL-10 did not affect the fusion process of preosteoclast-like cells to form osteoclast-like multinucleated cells in the rat coculture system of preosteoclast-like cells with primary osteoblasts. Furthermore, hIL-10 completely inhibited the colony formation induced by granulocyte macrophage colony-stimulating factor (GM-CSF). These findings suggest that the inhibition of osteoclastogenesis by hIL-10 started at the early stage of the differentiation of osteoclast progenitors to preosteoclasts.

Chromosome aberrations in cultured rat bone marrow cells treated with inorganic fluorides

The genotoxic effects of inorganic fluorides were investigated by treating cultured rat bone marrow cells with varying concentrations (0.1–100 μM) of potassium fluoride (KF) and sodium fluoride (NaF) for different durations (12, 24 and 36 h) and measuring the incidence of cells with aberrations and number of breaks per cell. Both forms of fluoride were found to be weak mutagens relative to the positive control N-methyl- N-nitro- N-nitrosoguanidine (MNNG). A specificity of fluoride ion in inducing chromosome aberrations (CA) was indicated by the observation that both NaF and KF behaved almost equivalently in this study and at significantly higher variations from the results with potassium chloride (KCl) and sodium chloride (NaCl).

Effect of BMP on the differentiation of bone marrow stromal cells into osteoblasts in a collagen-gel culture.

In-gel administration of recombinant BMP 2 (rBMP 2) to cultured bone marrow cells induces differentiation into osteoblasts much more efficiently than the conventional addition of rBMP 2 to the culture medium. We recently found that bone marrow cells cultured on collagen-gel could form bone-like calcified nodules withintwo weeks after confluence without the addition of the strong differentiationinducer dexamethasone. Using these cells on collagen-gel culture, the present studysought to evaluate the effects of rBMP 2 on cell differentiation. rBMP 2 was added to cultured rat bone marrow cells under three different conditions:(1) cellswere cultured in α-MEM with 15% fetal calf serum (standard medium) which contained various concentrations rBMP 2, (2) cells were cultured in standard medium which contained 10-8M dexamethasone (Dex), 10 mM β-glycerophosphate (β-GP), and rBMP 2 at different concentrations, and (3) cells were cultured on collagen-gel which contained rBMP 2 at different concentrations. In the standard medium (condition 1), the addition of rBMP 2 did not induce the cells to differentiate to form calcified nodules within a week, although both alkaline phosphatase activity and DNA content increased in a dose-dependent manner. In contrast, the addition of rBMP 2 as a component of collagen-gel (condition 3) markedly accelerated the phenotype expression of alkaline phosphatase and osteocalcin, as well as calcified nodule formation. This is the first report to demonstrate that collagen-gel enhances the effect of BMP in vitro. A similar effect wasobserved with rBMP 2 inthe presence of Dex and β-GP (condition 2).

Stromal components in rat bone marrow frozen sections compared to long-term rat bone marrow cultures

The haematopoietic microenvironment is believed to play an important role in controlling the haematopoietic process. Ultrastructural studies have shown that the haematopoietic stroma is composed of cellular as well as extracellular components. Relatively little is known about the distribution of the different stromal components in the bone marrow. The knowledge on the bone marrow microenvironment is mainly based on studies in which in vitro long-term bone marrow cultures have been used. Although this culture system offers a unique possibility to study haematopoietic in vitro, it does not fully represent the complexity of intact bone marrow. In the present study we describe the immunohistochemical distribution of different cellular and extracellular stromal components in frozen sections of rat bone marrow as well as in long-term bone marrow cultures, in order to compare the haematopoietic microenvironment used in in vitro studies in the in situ situation. We found that in situ a specific compartmentalization of stromal components exists in the bone marrow. Under culture conditions however, most stromal components are indeed present but the architecture present in the in situ situation had almost completely disappeared. The interaction between the different stromal elements was studied in the long-term bone marrow cultures. It appeared that under the chosen conditions, nodules were formed with a core of reticular cells and extracellular matrix. In close contact with this core immature macrophages appeared to proliferate and differentiate into mature, non-dividing macrophages.

Localization of osteopontin in resorption lacunae formed by osteoclast-like cells: a study by a novel monoclonal antibody which recognizes rat osteopontin

The characteristics of a monoclonal antibody produced against osteoclast-like multinucleated cells (MNCs) formed in rat bone marrow cultures were examined immunohistochemically and biochemically. The in vitro immunization was performed using as immunogen the MNCs from rat bone marrow cell culture, which revealed many characteristics of osteoclasts. After screening and cloning of hybridomas, the monoclonal antibody HOK 1 was obtained. This antibody reacted weakly with stromal cells and intensely with both MNCs and their putative migratory traces on culture dishes. Immunofluorescent examination of paraffin sections revealed intense reactivity on the epithelium of the choroid plexus, the ileum and the proximal-convoluted tubules of the kidney, and also on bone cells such as osteocytes, osteoblasts, and osteoclasts. Western blotting using purified rat osteopontin verified that the antigen recognized by HOK 1 was osteopontin. Positive HOK 1 immunoreactivity was further observed in the resorption lacunae formed by a culture of MNCs on human tooth slices and on the surface of osteoclasts. The present data suggested that osteopontin is preferentially present on the resorption lacunae in resorbing calcified matrices and that osteoclasts under a specific state might trap this protein on their cell surface.

A novel monoclonal antibody recognizing a unique antigen of rat osteoclasts induced by the calcified matrices.

The morphology of osteoclasts, primary cells that resorb bone, is well documented; however, the precise details of their terminal differentiation remains obscure. To date, the only morphological criterion for identifying activated functional osteoclasts has been the presence of ruffled borders. We have developed a rat bone marrow culture system in which osteoclast-like cells formed. These cells fulfilled most of the criteria of osteoclasts, and when they were reseeded on calcified tissue, formed numerous resorption lacunae in vitro. To find an immunological marker for functional osteoclasts, we have used these cells in a functional state as antigens for the preparation of monoclonal antibodies (mAb) that reacted with rat osteoclasts; we obtained mAb Ch1 and Ch2. Interestingly, these mAbs reacted with the marginal portion of authentic osteoclasts, where they attached to the bone surface on frozen sections. The reactivity of Ch1 to rat osteoclasts was more restricted than that of Ch2: Ch1 reacted with few tartrate-resistant acid phosphatase (TRAP)-positive cells on a culture plate. These TRAP-positive cells (including mono- and multinucleated cells) were, however, converted to Ch1-positive cells when they were reseeded on calcified tissues. These findings suggested that the antigen recognized by the Ch1 antibody was induced by some factors of matrix proteins released from calcified tissues.

The genotoxic and cytotoxic activities of inorganic fluoride in cultured rat bone marrow cells

The effects of sodium and potassium fluoride (NaF and KF) at concentrations ranging from 10(-7) to 10(-2) M for 12, 24, or 36 h on cultured rat bone marrow cells have been studied with respect to cytotoxicity and induction of sister-chromatid exchanges (SCE). At the three exposure times, cell survival progressively decreased with increasing concentrations. Treatment with 10(-2) M fluoride resulted in a statistically significant death (62-65%) of cells. Similarly, no dividing cells were encountered at concentrations of 10(-3) M and 10(-2) M, and significant reductions in mitotic index (MI) were calculated at 10(-4) M. In contrast, cell kinetics, expressed as cell proliferation index (CPI), revealed no significant inhibitory effect of fluoride on cell proliferation. Furthermore, the mean SCE score reached a maximum (7.64 SCE/cell) in the 24-h-treated cultures. This value was not significantly different from that observed in sodium chloride (NaCl) at 10(-2) M (5.42 SCE/cell) and distilled water (4.86 SCE/cell) controls. In comparison, mitomycin-C (MMC, positive control) at 5 x 10(-8) M caused an average of 22.13 SCE/cell. These results indicated an inhibition of cell division and death of cells with high doses of fluoride with no effect on SCE frequencies.

Macrophage colony-stimulating factor release and receptor expression in bone cells.

Colony-stimulating factors (CSF) may play a role in bone resorption. To examine whether osteoblasts secrete colony-stimulating activity (CSA) in response to parathyroid hormone (PTH) and parathyroid hormone-related peptide (PTHrP), conditioned medium (CM) from ROS 17/2.8 cells and primary rat osteoblasts were examined for induction of clonal growth of cultured rat bone marrow cells. Untreated cells constitutively secreted CSA, which increased with PTH and PTHrP treatment. The colonies formed were principally comprised of macrophages, and preincubation of CM with antiserum to murine macrophage colony-stimulating factor (M-CSF) neutralized most of the CSA, suggesting that the osteoblast-derived CSA was predominantly due to M-CSF. PTHrP treatment upregulated steady-state M-CSF mRNA levels. To investigate a paracrine role for M-CSF in bone we examined bone tissue and cells for the M-CSF receptor c-fms using immunohistochemical techniques and demonstrated staining of mature osteoclasts both in situ and after isolation. We conclude that M-CSF is responsible for the majority of the CSA released by PTH- and PTHrP-treated rat osteoblasts. In addition we identified CSF-1 receptor expression in mature osteoclasts. These data suggest that M-CSF is a mediator of osteoblast-osteoclast interaction in PTH- and PTHrP-induced bone resorption.

Heat-treated osteoblastic cell (ROS17/2.8)-conditioned medium induces the formation of osteoclast-like cells

To study the effects of osteoblast products on osteoclast formation, we added the conditioned medium (CM) of rat osteoblastic cell line ROS 17/2.8 to rat bone marrow cultures, in which tartrate-resistant acid phosphatase (TRAP)-positive osteoclast-like multinucleate cells (MNCs) formed in the presence of 10 −8 M 1,25-dihydroxyvitamin D 3 (1,25(OH) 2D 3). The formation of 1,25(OH) 2D 3-dependent TRAP-positive MNC at day 7 of culture was strongly inhibited by the >10 kDa fraction of ROS 17/2.8 cell-CM (ROSCM), but heat treated ROSCM (htROSCM) expressed marked stimulation in the formation of the MNCs. The expression of several osteoclastic phenotypes of the MNCs induced by htROSCM and 1,25(OH) 2D 3 was more enhanced compared with that of the MNCs induced by 1,25(OH) 2D 3 alone. The MNCs induced by htROSCM and 1,25(OH) 2D 3 were highly motile, were sensitive to calcitonin (CT), and had high bone resorbing activity. These data suggest that htROSCM promotes the osteoclast differentiation in the presence of 1,25(OH) 2D 3 in a rat bone marrow culture system. The stimulatory activity of TRAP-positive MNC formation in htROSCM is derived from heat-stable protein(s) that is (are) thought to be different from colony-stimulating factors (CSFs) such as macrophage-CSF (M-CSF) or granulocyte-macrophage-CSF (GM-CSF).

Induction of Mononuclear Precursor Cells with Osteoclastic Phenotypes in a Rat Bone Marrow Culture System Depleted of Stromal Cells

The importance of bone stromal cell involvement in osteoclast differentiation has been suggested. However, the detailed mechanism of its regulation is unclear. We investigated whether the soluble factors from osteoblastic cells influence osteoclast lineage cells in a population of rat bone marrow cells depleted of stromal cells. We show here that hematopoietic progenitor cells differentiate into mononuclear precursor cells, the preosteoclasts, that strongly express certain osteoclastic phenotypes in the absence of stromal cells. The multinucleation of the preosteoclasts occurred only in the presence of 1,25 dihydroxyvitamin D 3 and stromal cells. The preosteoclasts themselves did not have dentine-resorbing activity, but they could differentiate into multinucleate osteoclast-like cells having such activity in the presence of rat primary osteoblasts. This culture system is a unique differentiation system for preosteoclast induction.

Recombinant LD78 protein, a member of the small cytokine family, enhances osteoclast differentiation in rat bone marrow culture system

Cytokine LD78 is a member of the small inducible protein family involved in cell growth, wound healing and inflammation. However, its exact function is not known. In this study, we demonstrated that recombinant LD78 alpha and its variant LD78 beta proteins stimulate osteoclast-like cell formation in rat bone marrow cultures in the presence of 1 alpha, 25-dihydroxyvitamin D3. This enhancing activity was independent of prostaglandin synthesis. This is the first report which describes the effect of proteins involved in the small inducible protein family on the differentiation of osteoclasts.

12-O-tetradecanoylphorbol-13-acetate inhibits osteoclast-like cell differentiation in rat bone marrow cultures by inducing macrophage polykaryons

12-O-tetradecanoylphorbol-13-acetate inhibits osteoclast-like cell differentiation in rat bone marrow cultures by inducing macrophage polykaryons

Transforming growth factor beta inhibits megakaryocyte growth and endomitosis

Using a rat bone marrow culture system, the effect of transforming growth factor beta 1 (TGF beta 1) on megakaryocyte growth and endoreduplication has been studied. Purified human platelet TGF beta 1 inhibited the number of megakaryocytes that appeared in culture at a half-maximal concentration of 0.66 +/- 0.21 ng/mL and inhibited megakaryocyte endoreduplication at a half-maximal concentration of 0.14 +/- 0.08 ng/mL. Under identical conditions, growth of erythroid precursors was half-maximally inhibited at a concentration of 0.125 ng/mL while myeloid growth was not inhibited at concentrations of TGF beta 1 up to 25 ng/mL. These profound inhibitory effects on megakaryocyte growth and endomitosis suggested that TGF beta might play a role in megakaryocytopoiesis. Therefore, we explored the effect of TGF beta in three different experimental situations by using a neutralizing antibody to TGF beta: (1) Serum but not plasma was found to inhibit the number and ploidy of megakaryocytes that grew in vitro. This inhibitory activity was completely neutralized by antibody to TGF beta or on treatment with dithiothreitol. (2) Plasma from thrombocytotic rats was observed to decrease megakaryocyte ploidy on culture but this effect was not prevented by the addition of antibody to TGF beta. (3) Plasma from thrombocytopenic but not normal rats increased megakaryocyte ploidy on culture. Addition of antibody to TGF beta did not alter these results. Therefore, TGF beta is a potent inhibitor of the number and ploidy of megakaryocytes and accounts for all the inhibition seen when megakaryocytes are cultured in serum. However, the differences in effect on megakaryocyte growth that we observe between normal, thrombocytopenic, and thrombocytotic plasmas are not due to variations in the amount of TGF beta. Furthermore, our results show that release of TGF beta from megakaryocytes during culture does not act as an autocrine regulator of megakaryocyte ploidy in vitro.

Transforming Growth Factor β Inhibits Megakaryocyte Growth and Endomitosis

Using a rat bone marrow culture system, the effect of transforming growth factor beta 1 (TGF beta 1) on megakaryocyte growth and endoreduplication has been studied. Purified human platelet TGF beta 1 inhibited the number of megakaryocytes that appeared in culture at a half-maximal concentration of 0.66 +/- 0.21 ng/mL and inhibited megakaryocyte endoreduplication at a half-maximal concentration of 0.14 +/- 0.08 ng/mL. Under identical conditions, growth of erythroid precursors was half-maximally inhibited at a concentration of 0.125 ng/mL while myeloid growth was not inhibited at concentrations of TGF beta 1 up to 25 ng/mL. These profound inhibitory effects on megakaryocyte growth and endomitosis suggested that TGF beta might play a role in megakaryocytopoiesis. Therefore, we explored the effect of TGF beta in three different experimental situations by using a neutralizing antibody to TGF beta: (1) Serum but not plasma was found to inhibit the number and ploidy of megakaryocytes that grew in vitro. This inhibitory activity was completely neutralized by antibody to TGF beta or on treatment with dithiothreitol. (2) Plasma from thrombocytotic rats was observed to decrease megakaryocyte ploidy on culture but this effect was not prevented by the addition of antibody to TGF beta. (3) Plasma from thrombocytopenic but not normal rats increased megakaryocyte ploidy on culture. Addition of antibody to TGF beta did not alter these results. Therefore, TGF beta is a potent inhibitor of the number and ploidy of megakaryocytes and accounts for all the inhibition seen when megakaryocytes are cultured in serum. However, the differences in effect on megakaryocyte growth that we observe between normal, thrombocytopenic, and thrombocytotic plasmas are not due to variations in the amount of TGF beta. Furthermore, our results show that release of TGF beta from megakaryocytes during culture does not act as an autocrine regulator of megakaryocyte ploidy in vitro.

Trypsinized osteoclast-like multinucleated cells formed in rat bone marrow cultures efficiently form resorption lacunae on dentine

Rat bone marrow cultures containing 1α,25-dihydroxyvitamin D 3 [1α,25(OH) 2D 3] formed multinucleated cells (MNCs) that had many characteristics of osteoclasts. These MNCs, which have a tartrate-resistant acid phosphatase (TRAP) activity, could be classified into two morphological types: one type had smooth cellular margins (smooth-margined MNCs) and the other type had irregular spike-like margins (stellate MNCs). When bone marrow cells depleted of authentic osteoclasts were seeded and cultured on dentine slices, only low numbers of resorption lacunae could be detected. However, when preformed MNCs were detached by trypsinization and replated on dentine slices, numerous resorption lacunae were observed by scanning electron microscopy on these slices. Formation of lacunae occurred reproducibly during the five to ten days of culture. We also examined the effect of retinoic acid on TRAP-positive MNC formation in this bone marrow culture system. Although RA inhibited total TRAP-positive MNC formation, it increased the ratio of stellate MNCs to smooth-margined MNC, suggesting that RA may have the ability to regulate the formation of active osteoclasts.

Hematopoiesis on Suspended Nylon Screen-Stromal Cell Microenvironments

A three-dimensional culture system for the growth of primate and rodent bone marrow was developed in our laboratory. This method involves the seeding of stromal cells onto a nylon screen and the inoculation of fresh or cryopreserved bone marrow hematopoietic cells after stromal cell processes had extended across 3 to 4 out of every 5 mesh openings. Stromal cells attach, grow, and secrete matrix proteins which contribute to an intricate microenvironment for the support of multilineage hematopoiesis, which was observed for greater than 270 days in the rat model and for greater than 12 weeks in the human system, as evidenced by flow cytometry analysis and in vitro clonogenic assays. The adherent zones of these suspended nylon screen cultures consisted primarily of immature cells. These cultures could also be used as substrates for cytotoxicity measurements; treatment of rat bone marrow cultures of various ages with cytosine beta-D arabinofuranoside, cyclophosphamide, 5-fluorouracil, or methotrexate resulted in a dose-dependent decrease in CFU-C numbers and altered the phenotypic distribution of hematologic cells in the adherent zone. The use of a modification of this method to generate large numbers of active cytolytic cells after greater than 75 days culture of rat bone marrow-derived natural killer cells is described also. Suspended nylon screen bone marrow culture also has potential uses in genetic insertion and graft vs. host disease studies, blood component therapy, the evaluation of ex vivo purging programs, and in marrow expansion for transplantation.

Paradoxical regulation by PGE-2 on release of neutrophil chemoattractants by rat bone marrow macrophages

Prostaglandin E 2 (PGE 2) was shown to cause up to a 110% increase in the release into media of soluble chemoattractants for neutrophils by cultured rat bone marrow macrophages (RBMM) during a 16 hour incubation period. Coincubating with concentrations of PGE 2 of 10 nM and below did not stimulate release of chemoattractants while concentrations between 10 2 and 10 4 nM increased the chemoctactic activity of conditioned medium by 40% to 110% (p<0.05). In contrast to the effect of coincubating, pre-treatment with PGE 2 for 2 and 4 hours was ineffective in stimulating the release of chemoattractants by RBMM. We also assessed whether PGE-2 modulated the release of chemoattractants by RBMM stimulated ith endotoxin (LPS). LPS caused a four fold increase in the production of chemoattractants with a peak effect found at an LPS concentrations of 1 ug/ml. Coincubating with PGE 2 in concentrations between 10 2 and 10 4 nM paradoxically decreased LPS-stimulated production of chemoattractants by up to 40% (p<0.05). Pre-treatment with PGE 2 for 4 hours patially blocked LPS-stimulated release of chemotactic activity. These data indicate that PGE-2 has paradoxical effects on the production of chemoattractants by RBMM: being independently stimulatory but down regulating the effects of LPS. These findings suggest the possibility that the activation state of the RBMM may determine the effect of PGE 2: quiescent RBMM can be stimulated by PGE 2 but LPS-activated RBMM may be suppressed.

Stimulation of macrophage growth and multinucleated cell formation in rat bone marrow cultures by insulin-like growth factor I

In this study the effects of rhIGF-I on macrophage differentiation and growth have been studied using liquid suspension cultures of rat bone marrow cells. IGF-I stimulated macrophage growth in a dose-dependent manner, a maximum response was found at a concentration of 20 ng/ml. IGF-I effects could be ascribed to stimulation of both postmitotic and proliferating cells. A remarkable finding was that IGF-I induced formation of multinucleated cells (MNC). The MNC resembled macrophage-like cells (AcP, NSE positive). A monoclonal antibody to rhIGF-I significantly inhibited IGF-stimulated macrophage growth and MNC formation. A specific antibody to mouse CSF-1 reduced IGF-stimulated macrophage growth in mouse bone marrow cultures indicating that IGF-I effects could, at least in part, be ascribed to endogenous production of CSF-1. These findings indicate that IGF-I in concert with locally induced CSF-1 can influence the differentiation and growth of bone marrow-derived macrophages.