Inhibition of Osf2/Cbfa1 expression and terminal osteoblast differentiation by PPARgamma2.

Cells of the bone marrow stroma can reversibly convert among different phenotypes. Based on this and on evidence for a reciprocal relationship between osteoblastogenesis and adipogenesis, we have isolated several murine bone marrow-derived clonal cell lines with phenotypic characteristics of osteoblasts or adipocytes, or both. Consistent with a state of plasticity, cell lines with a mixed phenotype synthesized osteoblast markers like type I collagen, alkaline phosphatase, osteocalcin, as well as the adipocyte marker lipoprotein lipase, under basal conditions. In the presence of ascorbic acid and beta-glycerophosphate-agents that promote osteoblast differentiation-they formed a mineralized matrix. In the presence of isobutylmethylxanthine, hydrocortisone, and indomethacin-agents that promote adipocyte differentiation-they accumulated fat droplets, but failed to express adipsin and aP2, markers of terminally differentiated adipocytes. Furthermore, they were converted back to matrix mineralizing cells when the adipogenic stimuli were replaced with the osteoblastogenic ones. A prototypic cell line with mixed phenotype (UAMS-33) expressed Osf2/Cbfa1-a transcription factor required for osteoblast differentiation, but not PPARgamma2-a transcription factor required for terminal adipocyte differentiation. Stable transfection with a PPARgamma2 expression construct and activation with the thiazolidinedione BRL49653 stimulated aP2 and adipsin synthesis and fat accumulation, and simultaneously suppressed Osf2/Cbfa1, alpha1(I) procollagen, and osteocalcin synthesis. Moreover, it rendered the cells incapable of forming a mineralized matrix. These results strongly suggest that PPARgamma2 negatively regulates stromal cell plasticity by suppressing Osf2/Cbfa1 and osteoblast-like biosynthetic activity, while promoting terminal differentiation to adipocytes.

Effects of myelotoxic agents on cytokine production in murine long-term bone marrow cultures.

In long-term bone marrow cultures we studied the effect of the addition of the myelotoxic agents methotrexate (MTX) and ceftazidime (CEF) on the kinetics of cytokine production in the supernatant (SN) and on mRNA expression in the adherent stromal layer. In response to a medium change, a prompt and significant increase in colony-stimulating activity (CSA) and interleukin 6 (IL-6) concentrations in the SN occurred, peaking 12 h later. Two macrophage colony-stimulating factors (M-CSF) mRNA of 23 kb and 4 kb were identified. In response to the medium change, the 4.0-kb transcript increased significantly six h later. The 2.3-kb transcript expression was stronger than the 4-kb mRNA but did not cycle with medium change. At medium change, IL-6 mRNA was only minimally expressed; then a prompt increase occurred, which peaked six h later. The addition of 500 mg/ml (=915 microM) CEF to the culture caused a dose-dependent suppression of CSA and IL-6 supernatant concentrations and IL-6 and M-CSF mRNA expression. By contrast, 1 microM MTX had minimal effect on cytokine concentrations in the SN following medium change. mRNA expression was, however, suppressed. These results provide insights into the possible mechanisms whereby cytokines lead to increased myeloid cell proliferation following medium change. We also demonstrate that two myelotoxic agents have different effects on cytokine production. This information could be of value in developing rational approaches to the therapeutic use of cytokines in drug-induced neutropenia.

Upregulation of functional ryanodine receptors during in vitro aging of human diploid fibroblasts.

We demonstrate for the first time that cellular aging in vitro is accompanied by a dramatic elevation in the levels of ryanodine receptor-bearing Ca2+ channels. These channels normally reside within microsomal membranes and gate Ca2+ release from intracellular stores. We therefore measured cytosolic Ca2+ levels in 'young' (30 mean population doublings, MPDs) and 'senescent' (53 to 58 MPDs) human diploid fibroblasts (HDFs). Application of the known ryanodine receptor modulators, caffeine or cyclic adenosine diphosphate-ribose (cADPr), triggered cytosolic Ca2+ signals in both young and senescent cells. The signal magnitude however was significantly greater in senescent compared with young HDFs. In parallel, incubation with a highly specific anti-ryanodine receptor antiserum resulted in specific immunofluorescence only in senescent HDFs. We envisage that elevated levels of functional ryanodine receptors may underlie the defective Ca2+ handling and cellular degeneration that occurs with aging.

Impaired glutathione peroxidase activity accounts for the age-related accumulation of hydrogen peroxide in activated human neutrophils.

BACKGROUND: As assessed by flow cytometry, the increase in hydrogen peroxide in individual neutrophils from old volunteers was significantly greater than in neutrophils from young volunteers. To explain the discrepancy in previous reports that showed reduced superoxide generation with age and our finding, we measured the kinetics of antioxidative enzymes. METHODS: Neutrophils were obtained from young (ages 21-34) and old (ages over 65) volunteers. The increase in hydrogen peroxide following stimulation with formyl peptide in individual neutrophils was assessed by flow cytometry by using dihydrorhodamine 123. The enzyme kinetics was determined from the best fit curve using Michaelis-Menten equations. RESULTS: Aging was associated with a significant reduction in the Vmax for glutathione peroxidase. The decreased activity was not due to selenium deficiency as the serum and neutrophil concentrations were identical with age. Following activation, a significant increase in the Km was noted in neutrophils from young but not from old volunteers. CONCLUSIONS: These results account for the increased intracellular accumulation of hydrogen peroxide as a function of age in stimulated neutrophils. These results provide evidence in humans of an age-related impairment in antioxidative defense mechanisms that support the free radical theory of aging.

Cellular and molecular biomarkers indicate precocious in vitro senescence in fibroblasts from SAMP6 mice. Evidence supporting a murine model of premature senescence and osteopenia.

A variety of short-lived mouse strains (SAMP strains) and control strains of less abbreviated life span (SAMR strains) have been proposed as murine models of accelerated senescence. Each SAMP strain, in addition to displaying "progeroid" traits of accelerated aging, exhibits a singular age-related pathology. The application of this animal model to the study of normal aging processes has been and remains controversial. Therefore, we have undertaken a study of dermal fibroblasts derived from the short-lived SAMP6 strain, which shows early-onset and progressive osteopenia. We have investigated cellular and molecular characteristics that are associated with in vitro aging of normal human fibroblasts, and which are exacerbated in fibroblasts from patients with Werner syndrome, a human model of premature senescence. We found that SAMP6 dermal fibroblasts, relative to SAMR1 and C57BL/6 controls, exhibit characteristics of premature or accelerated cellular senescence with regard to in vitro life span, initial growth rate, and patterns of gene expression.

The pivotal role of interleukin 6 in formation and function of hematopoietically active murine long-term bone marrow cultures.

The multifunctional cytokine interleukin 6 (IL-6) is involved in the regulation of inflammatory and immune responses, and influences many bone and bone marrow functions. In this report we show high concentrations of IL-6 in the supernatant of murine long-term bone marrow cultures (LTBMC). The concentration increases following medium change peaking 12 h later. IL-6 plays a critical role in the generation and maintenance of myelopoiesis in LTBMC. The addition of monoclonal anti-IL-6 antibody to culture significantly suppresses myeloid cell production. IL-6 is also necessary for stromal layer development and the initiation of myelopoiesis in LTBMC. Horse sera (HS) containing low concentrations of IL-6 did not support LTBMC stromal layer development or myeloid cell production, whereas those with high concentrations did. LTBMC initially set up with horse serum containing high IL-6 concentration produced higher concentrations of colony-stimulating activity and IL-6 at the fifth week after culture initiation than those with low concentrations. The ability of a deficient serum to support myelopoiesis could be improved by the addition of recombinant IL-6 to culture. Similarly, the addition of an anti-IL-6 antibody to culture impaired the ability of a HS to initiate and support myelopoiesis in LTBMC. These results suggest that IL-6 is one of the factors that play an essential role in the formation and function of hematopoietically active LTBMC.

Increased adipogenesis and myelopoiesis in the bone marrow of SAMP6, a murine model of defective osteoblastogenesis and low turnover osteopenia.

Bone formation and hematopoiesis are anatomically juxtaposed and share common regulatory mechanisms. However, little is known about the interrelationship between these two processes. We have previously shown that the senescence accelerated mouse-P6 (SAMP6) exhibits decreased osteoblastogenesis in the bone marrow that is temporally linked with a low rate of bone formation and decreased bone mineral density. Here we report that in contrast to decreased osteoblastogenesis, ex vivo bone marrow cultures from SAMP6 mice exhibited an increase in the number of colony-forming unit adipocytes, as well as an increase in the number of fully differentiated marrow adipocytes, compared with SAMR1 (nonosteopenic) controls. Further, long-term bone marrow cultures from SAMP6 produced an adherent stromal layer more rapidly, generated significantly more myeloid progenitors and produced more IL-6 and colony-stimulating activity. Consistent with this, the number of myeloid cells in freshly isolated marrow from SAMP6 mice was increased, as was the number of granulocytes in peripheral blood. The evidence that SAMP6 mice exhibit decreased osteoblastogenesis, and increased adipogenesis and myelopoiesis, strongly suggests that a switch in the differentiation program of multipotential mesenchymal progenitors may underlie the abnormal phenotype manifested in the skeleton and other tissues of these animals. Moreover, these observations support the contention for the existence of a reciprocal relationship between osteoblastogenesis and adipogenesis that may explain the association of decreased bone formation and the resulting osteopenia with the increased adiposity of the marrow seen with advancing age in animals and humans.

Murine marrow stromal response to myelotoxic agents in vitro.

Previous studies have shown that the haemopoietically active murine long-term bone marrow culture (LTBMC) is a useful model for studying drug-induced suppression and recovery of myelopoiesis. We studied the effects on stromal morphology and stromal progenitors, assessed as colony forming unit-fibroblasts (CFU-F), of the addition of either the antimetabolite methotrexate (MTX) or the betalactam ceftazidime (CEF) to LTBMC. The examination of 500 micrograms/ml CEF-treated cultures revealed a stroma that appeared empty, with modest reduction in total stromal counts, and significant decreases in fat-containing and endothelial cells. In contrast, treatment with 1 microM MTX for 1 week initially caused minimal morphologic stromal changes, thereafter total stromal cell count as well as fibroblastoid, endothelial, fat containing and macrophage cells significantly increased. Haemopoiesis and the stroma recovered. Both CEF and MTX reversibly suppressed stromal progenitor cells in LTBMC. When added directly to CFU-F cultures, the concentrations resulting in a 50% colony growth inhibition were 214 micrograms/ml for CEF and 375 nM for MTX. These results suggest that CEF, but not MTX, has direct toxic effects on the stroma of established LTBMC. Stromal cell increase following MTX treatment probably indicates a stromal response that may contribute to haemopoietic cell recovery.

Excess formation of lysophosphatidic acid with age inhibits myristic acid-induced superoxide anion generation in intact human neutrophils.

A superoxide anion generation rate upon exposure to myristate of 1.93 +/- 0.34 nmol/min/10(6) cells in neutrophils from elderly human donors was significantly less than a value of 3.02 +/- 0.48 nmol/min/10(6) neutrophils from young donors. Myristate activation resulted in equal increases of AA in both the young and the old indicating no effect of aging on the PLA2 pathway to response. By contrast, the PLD-induced generation of PA was significantly higher in the old than in the young. In addition, myristate induced a significant age-related enhancement in LPA generation, in the old but not in the young. The mass of LPA generated following activation was 3.5 nmol/ 2.5 x 10(7) cells/ml in the young while in the old it averaged 7.0 nmol/2.5 x 10(7) cells/ml. The inhibitory effects of LPA may explain the age-related impaired ability to generate superoxide anion following activation by myristate.

Evidence suggesting a stimulatory role for interleukin-10 in erythropoiesis in vitro.

Interleukin-10 (IL-10) has been shown to exert anti-inflammatory effects by suppressing macrophage proliferation and inhibiting cytokine production. In this study we show that in the presence of erythropoietin (EPO), the addition of IL-10 results in a significant dose-dependent increase in both Burst Forming Unit-Erythroid (BFU-E) and Colony Forming Unit-Erythroid (CFU-E) colony growth in both serum-containing and serum-free murine cultures in vitro. IL-10 acts at the later stages of erythroid cell proliferation and differentiation as the increase in colony number was greater in CFU-E than in BFU-E, and was similar when IL-10 was added to BFU-E cultures at the time of culture initiation as when its addition to culture was delayed for 7 days. Furthermore, no increase in BFU-E colony number was noted when IL-10, added at the time of culture initiation, was neutralized by the addition to culture of a monoclonal anti-IL-10 antibody up to 7 days later. The increases in BFU-E by IL-10 addition were not the result of prolongation of BFU-E colony lifespan, which was not significantly different in IL-10 treated and control cultures, respectively. Rather IL-10 stimulated the proliferation of erythroid clusters that were now large enough to be recognized as colonies. IL-10-induced stimulation of erythropoiesis appeared to be independent of its inhibitory effects on macrophage function, as stimulation of erythroid colony growth was similar in macrophage-containing and depleted cultures. Studies to determine if the IL-10 effect was direct or indirect yielded equivocal results. A limiting dilution assay suggested a direct effect. However, a log/log dose response curve with IL-10 did not pass through the origin suggesting an indirect effect. These studies indicate that IL-10 acts synergistically with EPO to significantly increase stimulation of erythroid differentiation and proliferation in vitro and may be involved in the regulation of normal erythropoiesis in vivo.

Effect of age on the fatty acid composition of phospholipids in human lymphocytes.

We have examined the fatty acid composition of phospholipids of unstimulated and PHA-stimulated T cells from young and old donors. Our results demonstrate that aging is accompanied by decreases in the saturated fatty acids, myristic acid, and palmitic acid, and a concomitant increase in the unsaturated arachidonic acid. Following activation with PHA for 24 h, age-associated differences in fatty acids could no longer be detected. In contrast to the lymphocyte, aging did not affect the fatty acid composition of either serum or neutrophil phospholipids. Exposure of lymphocytes from old donors to myristic acid complexed medium increased the levels of myristate in the phospholipids to levels similar to that seen in lymphocytes from young donors. We conclude from these studies that aging is accompanied by an alteration in the fatty acid profiles of phospholipids, and that incubation in myristic acid complexed medium modulates these profiles. These alterations are unique to lymphocytes and may contribute to the age-related declines in lymphocyte function.

Approaches to the nutritional support of the older patient.

Malnutrition is a common problem in older persons, and in the presence of disease, is accompanied by increased morbidity and mortality. This article provides a rational approach to the nutritional care of the common nutritional problems that occur in the elderly. These problems include obesity, a condition that occurs with some prevalence in older persons, weight loss and being significantly underweight, and hypoalbuminemic malnutrition. An approach to the nutritional management of patients with pressure ulcers is also discussed.

Effect of age on marrow macrophage number and function.

Employing flow cytometry and a monoclonal antibody against the murine macrophage antigen, Mac-1, we found a significant increase in the number of marrow macrophages in aged mice. This was reflected as significant increase with age in the number of alpha-naphthyl acetate esterase positive cells, as well as in colony forming unit-macrophage (CFU-M) progenitor cells. Macrophages from the marrow of old mice generated significantly less tumor necrosis factor alpha (TNF alpha) than did macrophages from young mice, either spontaneously or when activated by granulocyte-macrophage colony stimulating factor (GM-CSF). Furthermore, conditioned medium (CM) derived from either marrow or peritoneal macrophages of old mice caused less suppression of burst forming unit-erythroid (BFU-E) colony growth than did CM obtained from young mice. Aging, therefore, is associated with an increase in the number of marrow macrophages that have an impaired ability to generate or release cytokines. The increase in macrophage number may reflect a compensation for their reduced function. Altered macrophage number and function may contribute to the age-related decline in hematopoietic reserve capacity.

Treatment of acute myelogenous leukemia in patients over 50 years of age with V-TAD: a Southwest Oncology Group study.

Acute myelogenous leukemia (AML) in the elderly continues to have a poor prognosis and new treatment approaches are needed. This Phase II trial was undertaken to evaluate the complete remission rate and toxicity of a chemotherapeutic regimen including etoposide and 6-thioguanine, combined with reduced doses of cytosine arabinoside and daunorubicin (V-TAD) in individuals greater than 50 years of age with AML. Thirty-five patients, ranging in age from 51 to 80 years (median, 66 years), were registered onto the study. Twenty-nine patients were entered at the first dose level (daunomycin 20 mg/m2 days 1 and 2, ara-C 75 mg/m2 days 1-5, 6-thioguanine 75 mg/m2 every 12 hr days 1-5, and etoposide 50 mg/m2 days 1, 2, and 3) and six patients underwent therapy at the second dose level (ara-C 75 mg/m2 days 1-7 with the remainder of the regimen unchanged). After achieving a complete remission, patients underwent two to three consolidation cycles of chemotherapy. Thirty-one patients were evaluable for response. Thirteen patients (ten of twenty-five at the first dose level and three of six at the second dose level) achieved a complete remission (42%). Median remission duration was 6 months (range 1-21 months). The current regimen, while tolerated, did not result in improved survival compared with prior treatment regimens because of a high incidence of resistant and recurrent leukemia.

Morphological characterization of stromal cell types in hematopoietically active long-term murine bone marrow cultures.

Accurate histological evaluation of stromal morphology is very difficult in cultures incubated in plastic flasks. Employing glass flasketts, we were able to characterize the morphology and immunocytochemistry of four marrow stromal cell types in a functionally intact microenvironment of murine long-term bone marrow cultures (LTBMCs). Fibroblastoid cells stained positively for collagen Type I and III, negatively for von Willebrand factor (vWf), the mouse macrophage F4/80 antigen, and the Bandeiraea simplicifolia lectin I isolectin B4 (BSL I-B4). Endothelial cells stained positively for vWf antigen and lectin BSL I-B4 but negatively for collagen Types I and III and for F4/80 antigen. Fat-containing cells had a dense, ovaloid, indented nucleus and fat-containing vacuoles. Macrophages were strongly positive for the F4/80 antigen and stained weakly with BSL I-B4. Between the fourth and ninth weeks after culture initiation, fibroblastoid and endothelial cells remained constant, between 21 +/- 2% and 24 +/- 2% and between 3 +/- 0.3% and 4 +/- 0.4%, respectively, of the total stromal cell population. By contrast, the percentage of fat-containing cells decreased significantly from 26 +/- 3% at Week 4 to 17 +/- 2% at Week 9, and macrophages increased significantly from 49 +/- 1% at Week 4 to 57 +/- 1% at Week 9. This characterization of the stromal cell types in functionally intact LTBMCs should assist in the study of the complex interactions among the marrow stroma, cytokine production, and hematopoiesis.

Interferon-gamma exerts its negative regulatory effect primarily on the earliest stages of murine erythroid progenitor cell development.

Interferon-gamma (INF-gamma) has been shown to suppress erythropoiesis and perhaps to contribute to the anemia of chronic disease. In this study we demonstrated that the concentration of INF gamma required to suppress murine burst forming unit-erythroid (BFU-E) growth was significantly less than that required to suppress colony forming unit-erythroid (CFU-E) growth. INF gamma acted at the most primitive step in erythroid progenitor cell differentiation and proliferation, as inhibition was maximal when added at the time of BFU-E culture initiation. Inhibition was progressively less if INF gamma addition was delayed after culture initiation. The effects of INF gamma on BFU-E did not require the presence of interleukin-1 alpha (IL-1 alpha), tumor necrosis factor-alpha (TNF alpha), or granulocyte macrophage colony stimulating factor (GM-CSF), as its effects were not neutralized by monoclonal antibodies against IL-1 alpha, TNF alpha, or GM-CSF. This applied whether INF gamma was added to culture with individual antibodies or with a combination of all three antibodies. INF gamma was not required for IL-1 alpha- or TNF alpha-induced suppression of BFU-E, as their effects were not neutralized by a monoclonal anti-INF gamma antibody. In contrast, GM-CSF-induced suppression of BFU-E was negated by the simultaneous addition of anti-INF gamma. We have previously shown that the addition of TNF alpha does not suppress BFU-E growth in cultures from marrow depleted of macrophages. Suppression did occur, however, if a small concentration of INF gamma that does not inhibit and increasing concentration of TNF alpha were added to culture, suggesting a synergistic effect between INF-gamma and TNF alpha. These observations suggest that INF gamma is a potent direct inhibitor of erythroid colony growth in vitro. It exerts its negative regulatory effect primarily on the earliest stages of erythroid progenitor cell differentiation and proliferation, as much higher doses are required to suppress late erythroid cell development. INF gamma is also involved in GM-CSF-induced inhibition of BFU-E colony growth.

Effects of ceftazidime, a betalactam antibiotic, on murine haemopoiesis in vitro.

Agranulocytosis has been reported in 5-15% of patients treated with high-dose betalactam antibiotics (BLA). We investigated the toxic effect of ceftazidime (CEF) as a representative of these antibiotics on colony-forming unit-granulocyte/macrophage (CFU-GM), on burst-forming unit-erythroid (BFU-E) colony growth and on myelopoiesis in murine long-term bone marrow culture (mLTBMC). The CEF concentration resulting in a 50% inhibition of growth was 146 micrograms/ml (267 microM) for CFU-GM, 132 micrograms/ml (241 microM) for BFU-E and 180 micrograms/ml (329 microM) for myeloid cell production in the supernatant of mLTBMC. Following addition of CEF to mLTBMC, CFU-GM remained low for 1 week and total myeloid cell production remained low for 2 weeks after removal of CEF from culture. Thereafter the values returned to control levels. The myeloid differential counts in the supernatant and adherent layers demonstrated a 'maturation arrest', which could be overcome by simultaneously adding all-trans retinoic acid to culture. These results demonstrate that CEF has reversible inhibitory effects on myelopoiesis and highlight the utility of in vitro haemopoietic assays as models to examine drug-induced haemopoietic dyscrasias.

Evidence suggesting a negative regulatory role for macrophages in murine erythropoiesis in vivo.

Increasing the rate of erythropoiesis in C57BL/6 mice, either by hypoxia or by the injection of recombinant erythropoietin (Epo), resulted in significant reductions in marrow macrophage number, as assessed by flow cytometry employing the monoclonal antibody against the macrophage antigen Mac-1 and by histologic determination of reductions in the number of marrow esterase-positive cells. This decline was paralleled by decreases in marrow colony-forming unit-macrophage (CFU-M) and colony-forming unit-granulocyte/macrophage (CFU-GM) number. The intramedullary concentration of the cytokines interleukin-1 alpha (IL-1 alpha) and tumor necrosis factor-alpha (TNF-alpha), which are produced by macrophages, was also reduced. Cessation of erythropoiesis was associated with increases in macrophage number, CFU-M and CFU-GM colony number, and IL-1 alpha concentrations. Increased erythropoiesis resulted in reductions in number of burst-forming unit-erythroid (BFU-E) colonies, which were less sensitive to suppression by macrophages as evidence by less increase in colony number when macrophages were removed from the marrow before in vitro BFU-E culture. BFU-E colony number was suppressed less when IL-1 alpha and TNF-alpha were added to cultures obtained from animals with stimulated erythropoiesis. Compared to controls, BFU-E number and suppression by macrophages increased significantly when erythropoiesis was reduced. These observations provide compelling evidence for a regulatory role for macrophages in normal erythropoiesis in vivo, presumably acting as a negative balance to the stimulatory effects of Epo.