Melphalan exposure induces an interleukin-6 deficit in bone marrow stromal cells and osteoblasts

Bone marrow stromal cells (BMSC) and osteoblasts are critical components of the microenvironment that support hematopoietic recovery following bone marrow transplantation. Aggressive chemotherapy not only affects tumor cells, but also influences additional structural and functional components of the microenvironment. Successful reconstitution of hematopoiesis following stem cell or bone marrow transplantation after aggressive chemotherapy is dependent upon components of the microenvironment maintaining their supportive function. This includes secretion of soluble factors and expression of cellular adhesion molecules that impact on development of hematopoietic cells. In the current study, we investigated the effects of chemotherapy treatment on BMSC and human osteoblast (HOB) expression of interleukin-6 (IL-6) as one regulatory factor. IL-6 is a pleiotropic cytokine which has diverse effects on hematopoietic cell development. In the current study we demonstrate that exposure of BMSC or HOB to melphalan leads to decreases in IL-6 protein expression. Decreased IL-6 protein is the most pronounced following melphalan exposure compared to several other chemotherapeutic agents tested. We also observed that melphalan decreased IL-6 mRNA in both BMSC and HOB. Finally, using a model of BMSC or HOB co-cultured with myeloma cells exposed to melphalan, we observed that IL-6 protein was also decreased, consistent with treatment of adherent cells alone. Collectively, these observations are of dual significance. First, suggesting that chemotherapy induced IL-6 deficits in the bone marrow occur which may result in defective hematopoietic support of early progenitor cells. In contrast, the decrease in IL-6 protein may be a beneficial mechanism by which melphalan acts as a valuable therapeutic agent for treatment of multiple myeloma, where IL-6 present in the bone marrow acts as a proliferative factor and contributes to disease progression. Taken together, these data emphasize the responsiveness of the microenvironment to diverse stress that is important to consider in therapeutic settings.     

The roles of protein tyrosine phosphatases in bone-resorbing osteoclasts

Maintaining the proper balance between osteoblast-mediated production of bone and its degradation by osteoclasts is essential for health. Osteoclasts are giant phagocytic cells that are formed by fusion of monocyte-macrophage precursor cells; mature osteoclasts adhere to bone tightly and secrete protons and proteases that degrade its matrix. Phosphorylation of tyrosine residues in proteins, which is regulated by the biochemically-antagonistic activities of protein tyrosine kinases and protein tyrosine phosphatases (PTPs), is central in regulating the production of osteoclasts and their bone-resorbing activity. Here we review the roles of individual PTPs of the classical and dual-specificity sub-families that are known to support these processes (SHP2, cyt-PTPe, PTPRO, PTP-PEST, CD45) or to inhibit them (SHP1, PTEN, MKP1). Characterizing the functions of PTPs in osteoclasts is essential for complete molecular level understanding of bone resorption and for designing novel therapeutic approaches for treating bone disease.     

Interleukin 1 induces ferritin heavy chain in human muscle cells

Interleukin 1 alpha (IL-1) and tumor necrosis factor alpha (TNF) are two monokines which play a prominent role in the response to inflammation and injury. We recently observed that TNF leads to an increase in the synthesis of the heavy chain of ferritin, suggesting that TNF may be involved in iron homeostasis (Torti et al. (1988) J. Biol. Chem. 263, 12638-12644). The experiments reported here demonstrate that in cultured human muscle cells, IL-1 induces ferritin H mRNA and protein as effectively as TNF. TNF and IL-1 were additive in their effects on ferritin H expression, and IL-1 induction of ferritin H was not blocked by anti-TNF antibodies. Ferritin H induction was a specific response not observed with beta or gamma interferon, nor with transforming growth factor beta. Both differentiated myotubes as well as myoblasts responded to IL-1 with the induction of ferritin H. These results suggest that monokine-mediated alterations in the subunit composition of the ferritin molecule may be of biological relevance in the response to inflammation and injury.     

ATPase pumps in osteoclasts and osteoblasts

Osteoblasts, osteocytes and osteoclasts are specialised cells of bone that play crucial roles in the formation, maintenance and resorption of bone matrix. Bone formation and resorption critically depend on optimal intracellular calcium and phosphate homeostasis and on the expression and activity of plasma membrane transport systems in all three cell types. Osteotropic agents, mechanical stimulation and intracellular pH are important parameters that determine the fate of bone matrix and influence the activity, expression, regulation and cell surface abundance of plasma membrane transport systems. In this paper the role of ATPase pumps is reviewed in the context of their expression in bone cells, their contribution to ion homeostasis and their relation to other transport systems regulating bone turnover.     

Overexpression of SPINDLIN1 induces cellular senescence, multinucleation and apoptosis

Human or mouse Spindlin1 is expressed in various tissues and cells, but its biological functions are poorly understood. In this study, we show that human SPINDLIN1 is localized to interphase nucleus and mitotic chromosomes, and its expression in HeLa cells is not regulated in a cell cycle-dependent manner. When SPINDLIN1 is stably overexpressed in HeLa cells, it results in multinucleation of cells, and these multinucleated cells exhibits characteristic features of senescence and apoptosis shown by growth and morphological alterations, beta-galactosidase activity, and Annexin V/7-Aminoactinomycin D staining. Mouse Spindlin1 is highly homologous with human Spindlin1, when overexpressed in NIH3T3 cells, it also induces multinucleation, senescence and apoptosis in murine cells. Our results demonstrate that SPINDLIN1 is an important gene for mammalian mitotic chromosome functions, and disrupted regulation results in abnormal cell division, a mechanism that may be involved in tumorigenesis.     

Osteoclastic activity induces osteomodulin expression in osteoblasts

Bone resorption by osteoclasts stimulates bone formation by osteoblasts. To isolate osteoblastic factors coupled with osteoclast activity, we performed microarray and cluster analysis of 8 tissues including bone, and found that among 10,490 genes, osteomodulin (OMD), an extracellular matrix keratan sulfate proteoglycan, was simultaneously induced with osteoclast-specific markers such as MMP9 and Acp5. OMD expression was detected in osteoblasts and upregulated during osteoblast maturation. OMD expression in osteoblasts was also detected immunohistochemically using a specific antibody against OMD. The immunoreactivity against OMD decreased in op/op mice, which lack functional macrophage colony stimulating factor (M-CSF) and are therefore defective in osteoclast formation, when compared to wild-type littermates. OMD expression in op/op mice was upregulated by M-CSF treatment. Since the M-CSF receptor c-Fms was not expressed in osteoblasts, it is likely that OMD is an osteoblast maturation marker that is induced by osteoclast activity.     

MKK6-p38 MAPK signaling pathway enhances survival but not bone-resorbing activity of osteoclasts

Phosphorylated p38 mitogen-activating kinase (MAPK) is observed in osteoclasts under in vivo inflammatory situations. However, the role of p38 MAPK in osteoclast function has not been elucidated, because all external stimuli tested hitherto failed to induce the phosphorylation of p38 MAPK in osteoclasts in culture. In this study, a constitutively active form of MKK6 (MKK6CA) was expressed in osteoclasts using adenoviral gene transfer in vitro. MKK6CA expressed in osteoclasts phosphorylated p38 MAPK and enhanced the survival of osteoclasts. Dentine-resorbing activity of osteoclasts was not enhanced by the MKK6CA expression. These results suggest that p38 MAPK signaling plays a critical role in the survival of osteoclasts in inflammatory diseases.     

IL-4 inhibits bone-resorbing activity of mature osteoclasts by affecting NF-kappa B and Ca2+ signaling

IL-4 is an important immune cytokine that regulates bone homeostasis. We investigated the molecular mechanism of IL-4 action on bone-resorbing mature osteoclasts. Using a highly purified population of mature osteoclasts, we show that IL-4 dose-dependently inhibits receptor activator of NF-kappaB ligand (RANKL)-induced bone resorption by mature osteoclasts. We detected the existence of IL-4R mRNA in mature osteoclasts. IL-4 decreases TRAP expression without affecting multinuclearity of osteoclasts, and inhibits actin ring formation and migration of osteoclasts. Interestingly, IL-4 inhibition of bone resorption occurs through prevention of RANKL-induced nuclear translocation of p65 NF-kappaB subunit, and intracellular Ca(2+) changes. Moreover, IL-4 rapidly decreases RANKL-stimulated ionized Ca(2+) levels in the blood, and mature osteoclasts in IL-4 knockout mice are sensitive to RANKL action to induce bone resorption and hypercalcemia. Furthermore, IL-4 inhibits bone resorption and actin ring formation by human mature osteoclasts. Thus, we reveal that IL-4 acts directly on mature osteoclasts and inhibits bone resorption by inhibiting NF-kappaB and Ca(2+) signaling.     

Normal human osteoclasts formed from peripheral blood monocytes express PTH type 1 receptors and are stimulated by PTH in the absence of osteoblasts

The prevailing view for many years has been that osteoclasts do not express parathyroid hormone (PTH) receptors and that PTH's effects on osteoclasts are mediated indirectly via osteoblasts. However, several recent reports suggest that osteoclasts express PTH receptors. In this study, we tested the hypothesis that human osteoclasts formed in vitro express functional PTH type 1 receptors (PTH1R). Peripheral blood monocytes (PBMC) were cultured on bone slices or plastic culture dishes with human recombinant RANK ligand (RANKL) and recombinant human macrophage colony-stimulating factor (M-CSF) for 16-21 days. This resulted in a mixed population of mono- and multi-nucleated cells, all of which stained positively for the human calcitonin receptor. The cells actively resorbed bone, as assessed by release of C-terminal telopeptide of type I collagen and the formation of abundant resorption pits. We obtained evidence for the presence of PTH1R in these cells by four independent techniques. First, using immunocytochemistry, positive staining for PTH1R was observed in both mono- and multi-nucleated cells intimately associated with resorption cavities. Second, PTH1R protein expression was demonstrated by Western blot analysis. Third, the cells expressed PTH1R mRNA at 21 days and treatment with 10(-7) M hPTH (1-34) reduced PTH1R mRNA expression by 35%. Finally, bone resorption was reproducibly increased by two to threefold when PTH (1-34) was added to the cultures. These findings provide strong support for a direct stimulatory action of PTH on human osteoclasts mediated by PTH1R. This suggests a dual regulatory mechanism, whereby PTH acts both directly on osteoclasts and also, indirectly, via osteoblasts.     

Dexamethasone inhibits bone resorption by indirectly inducing apoptosis of the bone-resorbing osteoclasts via the action of osteoblastic cells

Although glucocorticoids (GCs) are physiologically essentialfor bone metabolism, it is generally accepted that high dosesof GCs cause bone loss through a combination of decreased boneformation and increased bone resorption. However, the actionof GCs on mature osteoclasts remains contradictory. In thisstudy, we have examined the effect of GCs on osteoclasticbone-resorbing activity and osteoclast apoptosis, by using twodifferent cell types, rabbit unfractionated bone cells andhighly enriched mature osteoclasts (>95% of purity).Dexamethasone (Dex, 10^-10–10^-7 M) inhibited resorption pit formation on a dentine slice by the unfractionated bone cells in a dose- and time-dependent manner.However, Dex had no effect on the bone-resorbing activity of the isolated mature osteoclasts. When the isolated osteoclastswere co-cultured with rabbit osteoblastic cells, the osteoclastic bone resorption decreased in response to Dex,dependent on the number of osteoblastic cells. Like the effecton the bone resorption, Dex induced osteoclast apoptosis in cultures of the unfractionated bone cells, whereas it did not promote the apoptosis of the isolated osteoclasts. An inhibitorof caspases, Z-Asp-CH2-DCB attenuated both the inhibitory effecton osteoclastic bone resorption and the stimulatory effect onthe osteoclast apoptosis. In addition, the osteoblastic cellswere required for the osteoclast apoptosis induced by Dex. These findings indicate that the main target cells of GCs arenon-osteoclastic cells such as osteoblasts and that GCsindirectly inhibit bone resorption by inducing apoptosis ofthe mature osteoclasts through the action of non-osteoclasticcells. This study expands our knowledge about the multifunctional roles of GCs in bone metabolism.     

Bimodal actions of reactive oxygen species in the differentiation and bone-resorbing functions of osteoclasts

In order to demonstrate that cellular redox status undergoes decreased reduction during osteoclast differentiation and further decreased reduction during osteoclastic bone resorption, we analyzed gamma-glutamylcysteinyl synthetase activity, a glutathione synthesis rate-limiting enzyme, and total glutathione and thiol groups. Moderate and severe redox shifts towards a more oxidizing environment induced gradual increases and decreases in osteoclastogenesis. Moreover, while severe glutathione depletion inhibited bone resorption, moderate glutathione repletion enhanced bone resorption. In summary, our observations suggest that there is a threshold for redox status, representing biphasic patterns in osteoclast differentiation and function.     

Interleukin 2 induces proliferation of normal "resting" human T cells in the absence of other known external stimulation

Interleukin 2(IL-2) is known to stimulate the progression of activated T cells from G1 through the rest of the cell cycle. We have demonstrated that addition of purified recombinant human IL-2 (rIL-2) to fresh normal human peripheral blood mononuclear cells (PBM), which were IL-2 receptor (Tac) negative by FACS analysis, stimulated marked proliferation of the PBM. IL-2-induced proliferation was also observed with umbilical cord blood mononuclear cells. Monocyte depletion of PBM resulted in a marked reduction of rIL-2-induced proliferative response which could be restored by adding back autologous irradiated monocytes but not by interleukin 1. The T cells preincubated with rIL-2 showed a five to six times enhanced autologous mixed-lymphocyte reaction (AMLR) compared to controls. The rIL-2-induced proliferative response of PBM was inhibited in a concentration-dependent fashion by preincubation of PBM with an anti-HLA-DR framework monoclonal antibody. The proliferating cells were shown by two-color flow cytometric analysis to be primarily Leu-1+ and Leu-4+ T cells (both leu-3+ and Leu-2+ subsets); however, 6 to 19% of responding cells had surface markers for B cells or NK cells. The data demonstrate that rIL-2 can induce proliferation of "resting" human T cells. The phenomenon may be related to a monocyte-dependent AMLR which induces IL-2 receptors and IL-2 responsiveness in a subset of T cells.     

Effects of beta-adrenergic agonists on bone-resorbing activity in human osteoclast-like cells

In the present study, we demonstrate for the first time that beta-adrenergic agonists stimulate bone-resorbing activity in human osteoclast-like multinucleated cells (MNCs). Osteoclast-like MNCs constitutively expressed mRNA for alpha1B-, alpha2B- and beta2-adrenergic receptor (AR) in addition to characteristic markers of mature osteoclast, such as calcitonin receptor (CT-R), tartrate-resistant acid phosphatase (TRAP), alphaV-chain of integrin (Int alphaV), carbonic anhydrase II (CA-II) and cathepsin K (Cathe K). Epinephrine (1 microM; alpha,beta-adrenergic agonist) up-regulated expression of Int alphaV, CA-II and Cathe K in the osteoclast-like MNCs. Osteoclastic resorbing activity was markedly increased by isoprenaline (1 microM; beta-adrenergic agonist), moderately by epinephrine, but poorly by phenylephrine (1 microM; alpha1-adrenergic agonist). The actin ring, which was suggested to be correlated with bone-resorbing activity, was clearly observed in osteoclast-like MNCs treated with isoprenaline and epinephrine, but faintly in those treated with phenylephrine. These findings suggest that beta-adrenergic agonists directly stimulate bone-resorbing activity in matured osteoclasts.     

Interleukin 3 stimulation of tyrosine kinase activity in FDC-P1 cells

Interleukin 3 stimulates the proliferation of FDC-P1, a murine myeloid cell line, however the biochemical events subsequent to binding of IL3 have only recently begun to be investigated. We have previously described the activation of protein kinase C (PK-C) and serine/threonine phosphorylation of a 68 kd protein following IL3 treatment of FDC-P1 cells. Here we have used an anti-phosphotyrosine antibody to purify proteins containing phosphotyrosine following IL3 administration to FDC-P1 cells. We find that tyrosine phosphorylation of two proteins of 50 (pp50) and 70 (pp70) kilodaltons occurs rapidly following IL3 treatment. In addition to phosphotyrosine both proteins also contained phosphoserine. Together with previous evidence these results suggest that coactivation of serine/threonine and tyrosine kinase activities which target unique proteins may be an important element in IL3 signal transduction.     

Resorption-cycle-dependent polarization of mRNAs for different subunits of V-ATPase in bone-resorbing osteoclasts.

Protein sorting in eukaryotic cells is mainly done by specific targeting of polypeptides. The present evidence from oocytes, neurons, and some other polarized cells suggests that protein sorting can be further facilitated by concentrating mRNAs to their corresponding subcellular areas. However, very little is known about the mechanism(s) involved in mRNA targeting, or how widespread and dynamic such mRNA sorting might be. In this study, we have used an in vitro cell culture system, where large multinucleated osteoclasts undergo continuous structural and functional changes from polarized (resorbing) to a nonpolarized (resting) stage. We demonstrate here, using a nonradioactive in situ hybridization technique and confocal microscopy, that mRNAs for several vacuolar H(+)-ATPase subunits change their localization and polarity in osteoclasts according to the resorption cycle, whereas mRNA for cytoplasmic carbonic anhydrase II is found diffusely located throughout the osteoclast during the whole resorption cycle. Antisense RNA against the 16-kDa or 60-kDa V-ATPase subunit inhibits polarization of the osteoclasts, as determined by cytoskeleton staining. Antisense RNA against carbonic anhydrase II, however, has no such effect.     

Interleukin 2 induces interferon alpha/beta production in mouse bone marrow cells

We have previously reported that mouse bone marrow (BM) cells stimulated with alloantigen produce cytotoxic effector T-cell activity and produce interferon (IFN-)alpha/beta. In this report we show evidence suggesting that interleukin 2 (IL-2) may play a role in this IFN-alpha/beta production by alloantigen-stimulated BM cells. Alloantigen-induced IFN production by bone marrow cells was completely inhibited when cultures were supplemented with antisera to IL-2. Cell-free supernatants obtained at 2 days from cultures containing C57BL/6 BM cells and irradiated DBA/2J spleen cells were also shown to contain low levels of IL-2 activity and induced significant IFN production in fresh BM cells. Different IL-2 preparations were tested for their ability to induce IFN-alpha/beta production in mouse BM cells. Mouse BM cells cultured with recombinant human IL-2 or highly purified mouse IL-2 produced high levels of IFN-alpha/beta activity after 2-3 days of culture with significant IFN activity being detected as early as 24 hr of culture. IL-2-induced IFN-alpha/beta production was partially resistant to irradiation. In contrast, irradiated (2000 rad) bone marrow cells failed to produce any IFN when cultured with alloantigen in the absence of IL-2. T-cell-depleted BM cells or BM cells obtained from C57BL/10 nude mice produced high levels of IFN-alpha/beta following stimulation with IL-2. In addition, bone marrow cells depleted of Ia+, Qa 5+, or Asialo GM+1 cells produced IFN in response to IL-2. Thus, neither T cells nor NK cells are required for IL-2-induced IFN-alpha/beta production by BM cells. The action of IL-2 on bone marrow cells to induce IFN production was mediated by the classical IL-2 receptor, since monoclonal antibodies to the IL-2 receptor present on T cells blocked this response and since bone marrow cells depleted of IL-2 receptor-bearing cells failed to produce IFN when cultured with IL-2. These results suggest that non-T cells resident in the BM have receptors for IL-2 and can produce IFN-alpha/beta upon stimulation by IL-2. Since IFN has been shown to affect different aspects of hematopoiesis, the production of IFN by BM cells stimulated by IL-2 may be important in the control of hematopoiesis. In addition, IL-2-induced IFN production may play a role in graft-versus-host disease.     

白细胞介素1诱导激素对免疫应答的调节

在免疫应答中,由巨噬细胞产生的白细胞介素1(IL-1),不仅是介导免疫应答的一种重要因子,同时也是诱导激素对免疫应答进行反馈调节的一种重要媒介。胰岛素对免疫应答有促进作用,糖皮质激素对免疫应答有抑制作用;IL-1在低浓度时促进胰岛素分泌,在高浓度时则抑制胰岛素分泌,并诱导糖皮质激素产生。因此,不同浓度的IL-1可分别诱导激素对免疫应答的正负反馈调节,这种双向调节作用是维持免疫应答动态平衡的重要机制之一。