IL-6 stimulates osteoclast-like multinucleated cell formation in long term human marrow cultures by inducing IL-1 release

IL-6 enhances the differentiation of pluripotent hematopoietic stem cells but predominantly affects the differentiation of hematopoietic cells in the granulocyte-macrophage lineage. We have previously shown that multinucleated cells (MNC) with many features of the osteoclast phenotype form in long term human marrow cultures. Addition of rhIL-6 (10 to 100 pg/ml) to these cultures significantly increased MNC formation, with greater than 80% of the MNC expressing an Ag that cross-reacts with the mAb 23c6. This antibody preferentially binds to osteoclasts. rhIL-6 did not enhance MNC formation in marrow cultures treated with 1,25 dihydroxyvitamin D3, a potent stimulator of MNC formation, but significantly increased the percentage of MNC that cross-reacted with the 23c6 mAb. Addition of antihuman IL-1 to cultures treated with rhIL-6 totally inhibited the increase in MNC formation stimulated by rhIL-6. In contrast, anti-IL-1 did not affect MNC formation stimulated by 1,25 dihydroxyvitamin D3. Further, conditioned media from marrow cultures exposed to rhIL-6 contained elevated levels of IL-1 beta (500 pg/ml compared to 23 pg/ml in control cultures 15 h after IL-6 addition). These results suggest that the capacity of rhIL-6 to stimulate formation of MNC which cross-react with 23c6 is mediated by induction of release of IL-1 beta.     

The xid mutation affects hemopoiesis in long term cultures of murine bone marrow

Cells of the humoral immune system are particularly affected by a mutation at the X chromosome linked immunodeficiency disease (xid)locus. Although B cells are made in normal numbers, they fail to become phenotypically and functionally diverse. Consequently, poor antibody responses are mounted to certain types of Ag. There have been some indications that other types of hemopoietic cells may be influenced by the mutation and development of the humoral immune system is unusually dependent on the presence of T lymphocytes. We now describe an analysis of the lympho-hemopoietic environment studied with long term bone marrow cultures. Contrary to expectations, cultures initiated with cells from homozygous female or hemizygous male mice with the mutant allele established more quickly than normal. The accelerated initial growth pattern was clearly linked to the xid mutation. Artificial mixtures of marrow exhibited intermediate growth kinetics. Experiments with H-2 congenic and T6 chromosome marked cells did not reveal an intrinsic dominance of growth in nonadherent xid cells. Similar results were obtained with culture conditions which favored production of myeloid or lymphoid cells. These findings would be consistent with subtle changes in the bone marrow microenvironment resulting from the xid mutation. The pedigree of the mouse strains had a significant influence on lymphopoiesis in long term bone marrow cultures. Lymphocytes of BALB/c origin dominated over CBA/H background cells in cultures established from mixtures of the two, but this did not correlate with any functional deficiency in CBA/H stromal cells. In fact, establishment of an adherent layer was a rate-limiting step in initiating long term cultures and this could be achieved with a low dose inoculum of CBA/H marrow. Even more dramatic effects were found in hemopoietic cells from doubly defective C3H.nu/nu-xid mice. The bone marrow of these athymic animals contained normal numbers of granulocyte/macrophage progenitors. However, lymphoid cultures could not be reproducibly established with their cells and myelopoiesis was never observed in vitro. The relatively simple conditions which pertain in culture make it possible to appreciate effects of mutations and pedigree on hemopoiesis which are unremarkable in intact animals.     

Corticosteroid-resistant bone marrow-derived B lymphocyte progenitor for long term in vitro cultures

A long-term in vitro culture system derived from murine bone marrow cells can successfully support the growth of B cell precursors, pre-B cells, and IgM-expressing B cells. Intermediates in the B cell developmental pathway are known to have differential sensitivities to the toxic effects of corticosteroids. We demonstrate here that long term B lineage cultures can be established with the corticosteroid-resistant cell population from bone marrow. Kinetics for the establishment and growth of cultures derived from corticosteroid-treated marrow are similar to those observed with control cultures. Cells obtained from both sets of cultures have similar morphologies and ranges of phenotypic markers. These results indicate that the cell responsible for the outgrowth of the long term B lineage cultures is corticosteroid resistant and is likely to be earlier in the B lymphocyte lineage than steroid-sensitive pre-B cells.     

Abnormalities of B lineage cells are demonstrable in long term lymphoid bone marrow cultures of New Zealand black mice

The formation of B lymphocytes in young New Zealand Black (NZB) mice proceeds at an accelerated rate resulting in a deficiency of B lineage precursors in adult (greater than 15 wk old) animals. To study the characteristics of B lineage cells in young (4 wk) and old (6 mo) NZB mice, bone marrow from these animals was used to initiate long term lymphoid bone marrow cultures (LBMC) that permit the long term maintenance of B cells and their precursors. Age-matched cultures from BALB/c mice and NZB.xid marrow were established in parallel. Primary LBMC were readily established from these strains and showed similar patterns of growth for the 3-mo observation period. No significant differences in numbers of 14.8 positive cells were observed. However, NZB mice at both ages had a higher percentage of membrane IgM (mIgM)-expressing cells. Significant levels of supernatant IgM were found only in cultures of 6-mo NZB and BALB/c mice; levels were highest in NZB culture supernatants and were often more than 500 ng/ml; significant, although much lower, levels of IgG were likewise detected. Lymphoid cells from NZB.xid mice were unable to generate significant levels of IgM in supernatant fluids indicating the effects of the xid gene were displayed in vitro. Autoantibodies were not detected in any of the culture supernatants. Additional evidence for NZB hyperactivity in primary B lymphopoiesis was observed upon initiation of primary myeloid bone marrow cultures (MBMC) from these strains of mice and subsequently transferring them to LBMC conditions. This results in the cessation of myelopoiesis at the initiation of B lymphopoiesis. At the time of converting MBMC to LBMC, cultures of NZB and BALB/c mice morphologically resembled myeloid cultures and had neither B cell colony-forming units nor cells that expressed 14.8 or mIgM. However, following the switch, NZB mice had a 5-fold higher number of B cell colony-forming units. Further, MBMC established from NZB bone marrow cells had a reduced capacity to form colonies in the granulocyte-macrophage colony-forming unit assay. These studies indicate that defects of NZB hemopoietic cells are manifest in vitro and suggest the use of in vitro long term cultures as a valuable technique to further dissect the hematopoietic abnormalities of NZB mice and possible underlying microenvironmental defects.     

Nonrandom rearrangement of T cell receptor J alpha genes in bone marrow T cell differentiation cultures

TCR J alpha genes span a distance of approximately 65 kb on mouse chromosome 14. Due to the existence of 50 to 100 discrete J genes, a potential for great diversity exists within the V-J-C alpha gene products and within the ultimate repertoire of alpha beta TCR. We have prepared hybridomas from an in vitro system that supports T cell differentiation among bone marrow cells. We have examined the J alpha genes among these cells and categorized rearrangements according to their location within the J alpha locus. It was found that alpha rearrangements were always present among the hybridomas bearing beta gene rearrangements. When two bone marrow-derived alpha-bearing chromosomes could be demonstrated in these hybridomas, both were always rearranged and rearrangements on homologous chromosomes were shown to reside in similar regions of the J alpha locus. Most surprisingly, when hybridomas were categorized by the culture from which they derived, cells from the same culture (designated as a set) demonstrated a skewing of alpha rearrangements to restricted segments of J alpha genes. In one hybridoma, rearrangements on homologous chromosomes involved J alpha genes that were either identical or situated within a 1-kb segment of DNA. The skewing within sets could not be due to clonal identity between hybridomas as the beta and gamma rearrangements in all hybridomas were different. Results suggested that skewing of J alpha gene rearrangements occurred during the course of T cell development in vitro. Should the same situation occur in vivo, the number of distinct TCR J alpha sequences available for expression in early development may be far less than that predicted by gene number alone.     

In vitro differentiation of B lymphocytes from primitive hemopoietic precursors present in long-term bone marrow cultures

B lymphocytes are not produced in the Dexter long-term bone marrow cultures, but a primitive B cell precursor is present. The findings presented in this study demonstrate that this precursor can be induced to produce B lymphocytes by transferring the cultures to the Whitlock conditions for the long-term growth of B cells in vitro. Two weeks after the transfer of cultures maintained at 33 degrees C in medium supplemented with horse serum and steroids to low concentrations of fetal calf serum at 37 degrees C, marked effects can be observed. The pattern of cell growth changes from one in which the hemopoietic cells are clustered in tight foci containing several hundred cells to smaller ones in which the cells are not as densely packed. Fat cells in the adherent layer disappear and the supporting stroma becomes more uniform in appearance. This change in the culture format is accompanied by a decrease in the number of nonadherent cells and a shift from myelopoiesis to lymphopoiesis. The numbers of granulocyte-macrophage progenitors decline weekly after the change in culture conditions and are not detected by the third week. B cell colony-forming units appear by 3 wk. Cells that express the 14.8 cell surface antigen are induced by 1 wk after the change in culture conditions, followed by the appearance of surface IgM-bearing cells 2 wk later. This shift to lymphopoiesis can be confirmed morphologically. Granulocytes and macrophages disappear from the cultures by 4 wk, at which time almost all of the cells have a characteristic lymphocyte morphology. Upon switching these cultures back to the original Dexter conditions, only low levels of transient myelopoiesis can be reinitiated.     

Simvastatin induces osteoblastic differentiation and inhibits adipocytic differentiation in mouse bone marrow stromal cells

To clarify the mechanism of the stimulatory effect of statins on bone formation, we investigated the effect of simvastatin, a widely used statin, on osteoblastic and adipocytic differentiation in primary cultured mouse bone marrow stromal cells (BMSCs). Simvastatin treatment enhanced the expression level of mRNA for osteocalcin and protein for osteocalcin and osteopontin, and increased alkaline phosphatase activity significantly (p<0.05). After BMSCs were exposed to an adipocyte differentiation agonist, Oil Red O staining, fluorescence activated cell sorting, and decreased expression level of lipoprotein lipase mRNA showed that treatment with simvastatin significantly inhibits adipocytic differentiation compared to controls that did not receive simvastatin (p<0.05). Lastly, we found that simvastatin induces high expression of BMP(2) in BMSCs. These observations suggested that simvastatin acts on BMSCs to enhance osteoblastic differentiation and inhibits adipocytic differentiation; this effect is at least partially mediated by inducing BMP(2) expression in BMSCs.     

Effects of cyclic pressure on bone marrow cell cultures

The present in-vitro study used bone marrow cell cultures and investigated the effects of cyclic pressure on osteoclastic bone resorption. Compared to control (cells maintained under static conditions), the number of tartrate resistant acid phosphatase (TRAP)-positive, osteoclastic cells was significantly (p<0.05) lower when, immediately upon harvesting, bone marrow cells were exposed to cyclic pressure (10-40 kPa at 1.0 Hz). In contrast, once precursors in bone marrow cells differentiated into osteoclastic cells under static culture conditions for 7 days, subsequent exposure to the cyclic pressure of interest to the present study did not affect the number of osteoclastic cells. Most important, exposure of bone marrow cells to cyclic pressure for 1 h daily for 7 consecutive days resulted in significantly (p<0.05) lower osteoclastic bone resorption and in lowered mRNA expression for interleukin-1 (IL-1) and tumor necrosisfactor-a (TNF-a), cytokines that are known activators of osteoclast function. In addition to unique contributions to osteoclast physiology, the present study provided new evidence of a correlation between mechanical loading and bone homeostasis as well as insight into the molecular mechanisms of bone adaptation to mechanical loading, namely cytokine-mediated control of osteoclast functions.     

Human recombinant IL-3 stimulates B cell differentiation

To investigate the role of human IL-3 in B cell differentiation, we examined its effect on IgG secretion from normal B cells and a B cell line, JDA. The effect of IL-3 was compared to that of IL-6. IL-3 stimulated IgG secretion from tonsil B cells or peripheral blood-derived B cells activated by Staphylococcus aureus Cowan I strain. This effect required the presence of IL-2. Neither B cell growth factor (BCGF) nor IFN-gamma replaced IL-2 in this function. IL-6 stimulated similar IgG secretion from tonsil B cells and also required the presence of IL-2. Moreover, the combination of IL-3 and IL-6 induced IgG secretion equivalent to that induced by either lymphokine. These data suggest that IL-3 and IL-6 might affect normal B cell differentiation by similar mechanism(s). The IL-3 effect on B cells appears to be caused by direct interaction with B cells because IL-3 induced a dose-dependent stimulation of IgG secretion from the JDA cells. This stimulation did not require the presence of IL-2. IL-6 displayed a similar effect on JDA cells and did not require IL-2. However, when IL-3 was combined with IL-6 a synergistic IgG secretion was observed in JDA cultures. These data suggest that IL-3 may potentiate the human immune response via stimulation of B cell differentiation and that its effect is dependent on the target B cell population, its stage of activation and/or maturation.     

Dexamethasone stimulates osteogenic differentiation in vertebral and femoral bone marrow cell cultures: Comparison of IGF-I gene expression

Osteoblast-like cell cultures have been established from the marrow of adult rat vertebrae. We have simultaneously examined the response to dexamethasone (dex) treatment in cultures of young adult female vertebral and femoral marrow cells. Alkaline phosphatase (AP) activity was analyzed as well as the expression of mRNAs for osteocalcin (OC) and insulin-like growth factor I (IGF-I). The vertebral and femoral marrow cells were maintained for 7 days in primary culture with or without 10⁻⁸ M dex and then 6 days in secondary culture without dex or with 10⁻⁸ M or 10⁻⁷ M dex. All cells were examined on day 6 of secondary culture. Vertebral and femoral cultures each expressed the highest AP enzyme levels when grown with dex in primary culture (10⁻⁸ M) and secondary culture (10⁻⁷ M). Under all experimental conditions, vertebral cultures had lower AP enzyme activity than femoral cultures. When dex was omitted from secondary culture, OC gene expression was not detected in either vertebral or femoral passaged cells even if dex was present in primary culture. For dex conditions where OC was expressed, vertebral cultures had higher OC mRNA steady-state levels than femoral cultures. IGF-I gene expression was detected by Northern analysis in both vertebral and femoral secondary cultures. However, vertebral marrow cultures had much higher IGF-I mRNA levels compared to femoral cultures whether or not dex was present in primary culture. These findings demonstrate that dex supports the differentiation of both vertebral and femoral adult marrow osteogenic cells into osteoblasts. Our results support the hypothesis that osteoblastic marrow cultures differ depending upon which location in the skeleton they are from and that there are skeletal site–dependent differences in the insulin-like growth factor system components. J. Cell. Biochem. 71:382–391, 1998. © 1998 Wiley-Liss, Inc.     

Proliferation and differentiation of normal granulopoietic cells in continuous bone marrow cultures.

Modified conditions are reported for successful continuous bone marrow cultures with stem cell self-renewal and granulocyte-macrophage differentiation. Cells cultured over several weeks were found to be identical to freshly isolated bone marrow cells. Polymorphic neutrophils derived from cultures and primary bone marrow neutrophils both showed C3 AND IgG receptors and both actively phagocytosed foreign particles. Cultured and normal CFU-c were identical, both in their dose responsiveness to CFS and in their sedimentation rate characteristics.     

CARM1 deficiency inhibits osteoblastic differentiation of bone marrow mesenchymal stem cells and delays osteogenesis in mice

Bone repair remains a clinical challenge due to low osteogenic capacity. Coactivator associated arginine methyltransferase 1 (CARM1) is a protein arginine methyltransferase that mediates arginine methylation and endochondral ossification. However, the roles of CARM1 in osteoblastic differentiation and bone remodeling have not been explored. In our study, heterozygous CARM1-knockout (KO) mice were generated using the CRISPR-Cas9 system and a model of femoral defect was created. At day 7 postsurgery, CARM1-KO mice exhibited obvious bone loss compared with wild type (WT) mice, as evidenced by reduced bone mineral density (BMD), bone volume/total volume (BV/TV), trabecular thickness (Tb.Th), and trabecular number (Tb.N), and increased trabecular separation (Tb.Sp). Deletion of CARM1 in mice lowered synthesis and accumulation of collagen at the injury sites. The alkaline phosphatase (ALP) activity and osteogenic-related gene expression were declined in CARM1-KO mice. To further understand the role of CARM1 in osteoblastic differentiation, bone marrow mesenchymal stem cells (BMSCs) were isolated from the tibia and femur of WT or CARM1-KO mice. CARM1 deletion decreased histone arginine methylation and inhibited osteoblastic differentiation and mineralization. The mRNA sequencing of CARM1-KO BMSCs revealed the possible regulatory molecules by CARM1, which could deepen our understanding of CARM1 regulatory mechanisms. These data could be of interest to basic researchers and provide the direction for future research into bone-related disorders.     

Osteocalcin promotes differentiation of osteoclast progenitors from murine long-term bone marrow cultures

Murine long-term bone marrow cultures (LTBMCs) were used to generate hematopoietic cells free from marrow stromal cells. These progenitor cells were treated with GM-CSF (5 U/ml) with or without rat bone osteocalcin or rat serum albumin in either α-MEM with 2% heat-inactivated horse serum alone (α) or supplemented with 10% L-cell-conditioned medium (as a source of M-CSF) (L10). Few substrate-attached cells survived in basal α medium, but when treated with L10 medium or GM-CSF, they survived and proliferated. Osteocalcin did not significantly affect survival or proliferation. Subcultures of cells treated with GM-CSF had large numbers of multinucleated cells, more than half of which were tartrate-resistant acid phosphatase–positive (TRAP). Osteocalcin further promoted the development of TRAP-positive multinucleated cells; a dose of 0.7 μg/ml osteocalcin promoted osteoclastic differentiation by 60%. Using a novel microphotometric assay, we detected significantly more tartrate-resistant acid phosphatase activity in the osteocalcin plus GM-CSF group (75.6 ± 14.2) than in GM-CSF alone (53.3 ± 7.3). In the absence of M-CSF, GM-CSF stimulated tartrate-resistant acid phosphatase activity, but osteocalcin did not have an additional effect. These studies indicate that osteocalcin promotes osteoclastic differentiation of a stromal-free subpopulation of hematopoietic progenitors in the presence of GM-CSF and L-cell-conditioned medium. These results are consistent with the hypothesis that this bone-matrix constituent plays a role in bone resorption. © 1994 Wiley-Liss, Inc.     

Renewal of hematopoietic stem cell clones in long-term bone marrow cultures

In long-term cultures of murine bone marrow, clonal succession of hemopoietic cells was observed as measured by karyologic analysis. There were high oscillations in self-renewal of CFUs in the cultures. A close correlation between the CFUmix karyotype and mitotic non-adherent cells in culture (but not between these cell types and CFUs) was revealed.     

Messenger RNA turnover during bone marrow erythroid cell differentiation

Incubation of bone marrow cells from anaemic rabbits in the presence of actinomycin D led to a decrease in total protein synthesis and an increase in the relative synthesis of globin. This increase in the proportion of globin was observed with in vivo labelling of cellular proteins and in vitro translation of isolated RNA, which indicates that the messenger RNA for globin is much more stable than the other bone marrow cell messages. This was further shown by pulse-labelling the RNA and characterization of the different species by separation on a cDNA-oligo(dT)-cellulose column. Within 12 h after pulse-labelling the relative levels of globin mRNA had risen 10-fold, while a rapid decrease in the level of the poly(A)-rich RNA fraction was observed. Investigations into the mechanisms of this differential stability indicate that the more metabolically active cells from the early stages of erythropoietic development are more susceptible to inhibitors of RNA synthesis such as actinomycin D and alpha-amanitin. A preliminary study using a lysosomal inhibitor, chloroquine, indicates that there appear to be at least two degradative mechanisms, involving a lysosomal and a non-lysosomal pathway, with selective specificity for different messages.     

Characterization of early B lymphocyte precursors present in long-term bone marrow cultures

Lymphoid precursor cells are present in long-term bone marrow cultures (LTBMC), but their differentiation into mature lymphocytes is blocked. A quantitative assay for B cell precursors in LTBMC, which gives a linear relationship between the number of grafted LTBMC cells and the frequency of B cell colony forming units (CFU-B) in the spleen and bone marrow of immunodeficient CBA/N mice 19 days after reconstitution, is described. Characterization of the B cell precursor indicates that this assay is detecting a very early precursor and not a B lymphocyte or a late pre-B cell. This conclusion is based on the observations that a) pre-B cells transformable by Abelson murine leukemia virus are not present in LTBMC by 3 days postrecharge and CFU-B are absent by 6 days postrecharge; b) late B cell progenitors capable of rapid repopulation of irradiated CBA/N mice are not present in LTBMC, since a lag in the kinetics of B cell reconstitution in animals grafted with LTBMC cells is observed compared with fresh bone marrow cells; c) the B cell precursors in LTBMC have high proliferative potential, since they can stably repopulate recipient mice for at least 8 wk postreconstitution and through two serial passages in irradiated CBA/N recipients; and d) the B cell precursors are large, rapidly sedimenting cells as determined by velocity sedimentation. The serial transplantation experiment further shows that a split is often observed between lymphoid and myeloid reconstituting ability of LTBMC cells. The LTBMC B cell precursor may be a pluripotent stem cell or a lymphoid stem cell, although its differentiative potential remains to be determined.     

IL-3 induces differentiation of bone marrow precursor cells to osteoclast-like cells

IL-3, a cytokine with hematopoietic differentiating capability, induced murine bone marrow cells to differentiate into cells resembling osteoclasts. The cells resulting from treatment with IL-3 were multi-nucleated and demonstrated tartrate-resistant acid-phosphatase activity, as do resident osteoclasts found in bone. IL-3-induced osteoclast-like cell development in the absence of serum-derived vitamin D metabolites, and a mAb that inhibited IL-3-induced proliferation of an addicted cell line also inhibited the development of osteoclasts in the presence of IL-3. The same Ab had no effect on 1 alpha, 25-dihydroxyvitamin D3-induced differentiation of osteoclasts. This newly described function of IL-3 may indicate a role for activated T cells in the bone resorption seen with rheumatoid arthritis.     

A stromal cell line from myeloid long-term bone marrow cultures can support myelopoiesis and B lymphopoiesis

The production of B lymphocytes and myeloid cells occurs in the bone marrow in association with a supporting population of stromal cells. To determine whether these processes are dependent upon the same or different populations of stromal cells, stromal cell lines were generated from the adherent layer of a Dexter type long-term bone marrow culture. These cultures support myeloid cells and their precursors, a B cell precursor, and the adherent layer cells with support B cell differentiation under appropriate conditions. Two of the lines examined, S10 and S17, express class I histocompatibility antigens but not other hemopoietic cell surface determinants such as Thy-1, Lyt-1, Ig, Ia, Mac-1, or BP-1. Both lines could support myelopoiesis under Dexter conditions upon seeding with nylon wool-passed bone marrow. The nylon wool passage depletes stromal cells capable of forming adherent layers in vitro but retains hemopoietic precursors. The number of cells and colony-forming units-granulocytes/macrophages in the nonadherent cell population recovered 3 wk post-seeding had increased 19-fold and 10-fold, respectively, in the reseeded cultures of S10 and S17. After 3 wk of growth in Dexter conditions, the reseeded cultures were transferred to conditions optimal for B cell differentiation described by Whitlock and Witte. After 4 wk of growth, hemopoietic cells were consistently recovered from S17 cultures but not those of S10. A proportion of these cells from S17 cultures expressed the 14.8 antigen and were surface IgM positive. Surviving hemopoietic cells present in cultures of S10 were primarily macrophages. These findings indicate that S17 but not S10 can support both myelopoiesis and B lymphopoiesis and suggest that one stromal cell population has the capacity to form a hemopoietic microenvironment for both lineages.     

The role of IL-5 in IgA B cell differentiation

IL-5 enhances secretion of IgA by B cells. The stage of B cell differentiation at which IL-5 enhances IgA secretion and the mechanism by which it exerts this effect are unknown. We examined these issues by separating Peyer's patch (PP) B cells into membrane IgA (mIgA)-positive and mIgA-negative cells with panning or cell sorting. When LPS was used to activate these cells, mIgA-positive PP B cells were induced by IL-5 (either as crude T cell supernatant or rIL-5 to secrete large amounts of IgA. In contrast mIgA-negative PP B cells showed no significant amount of IgA secretion with IL-5. In addition, rIL-5 did not cause expression of mIgA by mIgM-bearing B cells. The mechanism involved in enhancement of IgA secretion was evaluated by utilizing an ELISPOT assay to quantitate IgA secreting cells. Both unsorted PP B cells and mIgA-positive PP B cells, when incubated with IL-5, showed an increase in the number of IgA-secreting cells that was proportional to the increase in total secreted IgA. However, LPS-activated PP mIgA-positive B cells, when incubated with rIL-5, showed no increase in proliferation, as measured by [3H]thymidine incorporation indicating that the increase in IgA-secreting cells after incubation with IL-5 occurred not as a result of proliferation but rather through promotion of terminal differentiation. Thus, IL-5 acts as a differentiation factor on B cells which have already undergone isotype switch to IgA B cells, promoting differentiation into IgA-secreting cells with resultant increased IgA secretion.     

Granulocyte colony-stimulating factor synergistically augments 1,25-dihydroxyvitamin D3-induced monocytic differentiation in murine bone marrow cell cultures.

In a series of studies, we have reported that 1,25-dihydroxyvitamin D (3), a known stimulator of monocytic differentiation, primes bone marrow progenitor cells or promyelocytic HL-60 cells to the actions of several factors involved in both monocytic and granulocytic differentiation. In the present study, we have further examined the combinational effects of 1,25-dihydroxyvitamin D (3) and the other inducer of granulopoiesis, granulocyte colony-stimulating factor, on non-fractionated native murine bone-marrow cell culture. Over 6 days of treatment, human granulocyte colony-stimulating factor sustained cell viability, increased the size of small rounded non-adherent cells, and induced granulocytic differentiation, while 1,25-dihydroxyvitamin D (3) decreased cell viability, promoted the development of large adherent flattened cells, and upregulated some monocytic differentiation markers. Combining these two factors over 6 days synergistically upregulated phagocyte activity, membrane-bound interleukin-1alpha, NAD(P)H oxidase, monocytic Mac-1, and non-specific esterase. Similar effects were observed in successive treatment with granulocyte colony-stimulating factor followed by 1,25-dihydroxyvitamin D (3), but successive treatment in reverse order was somewhat less effective. No combinational treatment upregulated granulocytic lactate dehydrogenase, Gr-1, or chloroacetate esterase to as great an extent as was obtained with granulocyte colony-stimulating factor alone, indicating that granulocytic differentiation is attenuated by addition of 1,25-dihydroxyvitamin D (3). Therefore, in contrast to our previous data, the present findings suggest that granulocyte colony-stimulating factor synergistically augments 1,25-dihydroxyvitamin D (3)-induced monocytic differentiation in our murine bone-marrow cell cultures. Considering previously published data, we also suggest that these synergistic effects may be mainly due to the combination of two distinct effects such as the primary proliferative effects of granulocyte colony-stimulating factor on multipotent stem cells and the subsequent differentiative effects of 1,25-dihydroxyvitamin D (3) on proliferating cells.