Preferential recognition of a fragment species of osteoarthritic synovial fluid fibronectin by antibodies to the alternatively spliced EIIIA segment.

OBJECTIVE: To characterize the species of synovial fluid (SF) fibronectin (FN) bearing the alternatively spliced EIIIA segment. METHODS: SF from patients with osteoarthritis (OA) and rheumatoid arthritis (RA), as well as corresponding affinity isolation products, were subjected to 1-dimensional and 2-dimensional electrophoresis followed by Western blot analysis. RESULTS: Regardless of the clinical type of arthritis, a polyclonal antibody that recognizes antigenic determinants throughout the FN molecule produced staining of predominantly approximately 200+ and approximately 170-kd species in reduced 1-dimensional electrophoresis. Despite the overall prevalence of the larger species, 4 monoclonal antibodies (mAb) reactive with sequences lying near the center of the EIIIA segment exhibited a relative failure to recognize the larger of these 2 species in OA, but not RA, SF. The absence of recognition of EIIIA sequences within the approximately 200+ kd forms of OA SF FN was unrelated to their derivation from dimers, since anti-EIIIA mAb recognized the smaller fragment species in preference to both monomeric and dimeric forms. The approximately 170-kd EIIIA+ fragments were observed to have minimal gelatin-binding capacity and appeared on 2-dimensional electrophoresis to extend from the N-terminus of FN through at least the center of the EIIIA segment. Similar results were obtained for samples obtained by needle aspiration or arthroscopic lavage, suggesting a widespread applicability of these findings. CONCLUSION: The approximately 170-kd EIIIA+ species of FN could potentially constitute a soluble "vehicle" by which chondrocyte-regulating EIIIA sequences, liberated from inhibitory flanking C-terminal sequences, could reach cells in the arthritic joint. Additionally, "FN species-specific" recognition of this segment within OA SF could constitute a marker by which to gauge the activity of the OA disease process.

Increased body weight in C57BL/6 female mice after exposure to ionizing radiation or 60Hz magnetic fields.

PURPOSE: The purpose of this investigation was to determine whether early treatment with ionizing radiation and/or chronic magnetic field (MF) exposure affected body weight in female mice. MATERIALS AND METHODS: Weanling C57BL/6 female mice were irradiated with four equal weekly cobalt-60 exposures (total cumulative doses: 3.0, 4.0, 5.1Gy) and/or received chronic lifetime exposure to 1.4 mT 60 Hz circularly polarized MF or ambient MF. The body weights of 2280 mice were recorded at 35 age intervals, and analysis of variance was used to compare the mean differences from baseline weights between treatment groups and sham-exposed controls. RESULTS: A highly statistically significant effect of ionizing radiation on body weight was observed at 28 age intervals (p < or = 0.001), and for MF exposure at 10 age intervals (p < or = 0.001). During the young adult growth phase, mice exposed only to MF exhibited < or =0.5 g greater weight gain relative to sham-exposed controls (p = 0.0001). The effect of ionizing radiation alone was inversely related to dose, with the largest weight increases observed in all of the irradiated groups after 9-12 months (p = 0.0001). CONCLUSIONS: Treatment with split-dose ionizing radiation at an early age and chronic exposure to a residential power frequency MF were found to produce small but significant increases in body weight.

Hematopoietic neoplasia in C57BL/6 mice exposed to split-dose ionizing radiation and circularly polarized 60 Hz magnetic fields.

This study assessed the effect of chronic exposure to a 60 Hz circularly polarized magnetic field on the occurrence of ionizing radiation-induced lymphoma and other hematopoietic neoplasia in mice. Female C57BL/6 mice received lifetime exposure to either a magnetic field flux density of 1.42 mT for 18 h/day, or an ambient magnetic field of 0.13 microT. Beginning on the first day of magnetic field exposure, 1710 mice were treated with one of three levels of split-dose Cobalt-60 gamma-radiation (cumulative 3.0, 4.0 or 5.1 Gy). The remaining 570 mice received sham irradiation treatment. Sections from 10 lymphoid tissues were evaluated histopathologically for hematopoietic neoplasia. The primary statistical analysis used the Poly3 method to compare lymphoma incidences in magnetic field (MF)-exposed and control mice. Secondary analyses used the Cox proportional hazards model to analyze incidence rates for mortality and development of specific types of neoplasia. The mortality incidence rate was increased by ionizing radiation treatment, and all neoplasms were observed sooner in irradiated mice. However, the lifetime incidence of hematopoietic neoplasia was similar in all experimental groups, including those that were not exposed to ionizing radiation. Chronic exposure to MFs did not affect the mortality incidence rates and did not change the relative incidences of hematopoietic neoplasia in mice that received the same ionizing radiation treatment, with the exception of a marginally significant reduced relative risk of 0.97 (P = 0.05) for lymphoblastic lymphoma in mice exposed to a magnetic field and treated with 5.1 Gy. Lymphomas and histiocytic sarcomas were first observed approximately 50 days sooner in mice that were exposed to magnetic fields but not ionizing radiation, although this comparison was not statistically significant and the incidence of hematopoietic neoplasia in these mice was not different from that of mice in the 0 T/0 Gy group.

Primary brain tumor incidence in mice exposed to split-dose ionizing radiation and circularly polarized 60 Hz magnetic fields.

Three sections of brain tissue from 2,657 female C57BL/6 mice were stained with hematoxylin and eosin and evaluated microscopically for proliferative lesions. Mice had been treated with either split-dose ionizing radiation (0, 3.0, 4.0 and 5.1 Gy), chronic lifetime 60 Hz circularly polarized magnetic field exposure (ambient or 14.2 G, 1.4 mT), or both, and were evaluated after natural death or euthanasia for humane reasons. Among 950 mice which were not treated with ionizing radiation, two benign spontaneous lipomatous hamartomas were observed. Seven primary brain tumors of various types not found in untreated animals were observed among the 1,707 irradiated mice. Possible promotional effects of magnetic field exposure on primary brain tumor development and incidence could not be assessed due to the low number of tumors observed.

Surface adhesion-mediated regulation of chondrocyte-specific gene expression in the nontransformed RCJ 3.1C5.18 rat chondrocyte cell line.

Recent evidence suggests that decreased chondrocyte function in osteoarthritis and other articular disorders may be due to chondrocyte dedifferentiation produced by altered regulatory signals from the cartilage extracellular matrix (ECM). However, there are currently no mammalian chondrocytic cell line systems adapted to the study of this process. We therefore examined the effects of ECM growth conditions on markers of differentiated chondrocytic phenotype expression in the nontransformed rat RCJ 3.1C5.18 (RCJ) chondrocyte cell line, including type II collagen expression, aggrecan production, link protein gene expression, and parathyroid hormone (PTH) receptor number. RCJ cells grown in monolayer on plastic exhibited a dedifferentiated phenotype characterized by flattened cell morphology, with > 80% type I collagen and < 5% type II collagen production, as determined by two-dimensional gel mapping electrophoresis of collagen cyanogen bromide peptides. In addition, aggrecan production was low, and link protein mRNA was not expressed at detectable levels. After transfer to growth under minimal attachment conditions on the surface of a composite type I collagen/agarose (0.15%-0.8%) gel (CAG) for 7 days, RCJ cells developed a rounded, chondrocytic morphology and a pattern of differentiated, chondrocytic gene expression, with 79% type II and 8% type I collagen production. Steady-state type I and type II procollagen mRNA levels were altered in parallel with collagen protein expression. In cells grown on CAG, aggrecan production increased 6-fold, and there was a marked increase in both aggrecan core protein and link protein mRNA levels. In addition, maximal PTH-stimulated cAMP generation increased 15-fold in association with an increased PTH receptor number. Therefore, the RCJ chondrocyte cell line is highly sensitive to ECM regulation of chondrocyte-specific gene expression.

Effects of prostaglandins on deoxyribonucleic acid and aggrecan synthesis in the RCJ 3.1C5.18 chondrocyte cell line: role of second messengers.

PGs play an important role in regulating articular chondrocyte function in both normal and pathological states. However, the mechanisms of the effects of PG on chondrocyte function remain undefined. We, therefore, examined the effects of PGE1, PGE2, and PGE2 alpha on second messenger generation in relation to DNA and aggrecan synthesis in the nontransformed rat RCJ 3.1C5.18 (RCJ) chondrocyte cell line. RCJ cells were grown under minimal attachment conditions on a composite collagen-agarose (0.15%/0.8%) gel to maintain a differentiated phenotype. PGE1 and PGE2 (0.001-100 microM) produced a similar dose-related increase in cAMP accumulation, with a maximal 8-fold increase over basal values, whereas PGF2 alpha produced a minimal 1.3-fold increase in cAMP levels only at 100 microM. On the other hand, both PGE2 and PGE2 alpha raised the intracellular free calcium ([Ca2+]i) concentration, derived primarily from extracellular sources, whereas PGE1 was without effect on [Ca2+]i. These three PGs also had divergent effects on DNA synthesis, as measured by [3H]thymidine ([3H]TdR) incorporation. PGF2 alpha (0.001-5 microM) produced a dose-related increase in [3H]TdR incorporation, with a maximal 1.6-fold increase over baseline values at 5 microM and a slight decline to below maximal levels at 10 microM. PGE2 exhibited a contrasting inverse biphasic response, with an initial small suppressive effect that was maximal at 0.1 microM and a secondary stimulatory phase producing a small increase over control values at 5 microM. PGE1 had a uniformly suppressive effect, producing a 30% decrease at 10 microM. Despite the divergent effects of PGE1, PGE2, and PGE2 alpha on second messenger generation and DNA synthesis, all three PGs produced a dose-related stimulation of aggrecan synthesis. PGF2 alpha was the most potent, producing significant stimulation at 0.001 microM and a maximal 104% increase at 5 microM. PGE1 and PGE2 were approximately equipotent and approximately 60% as effective as PGF2 alpha in stimulating aggrecan synthesis. Northern analysis demonstrated that the effects of PG on aggrecan synthesis were not accompanied by changes in aggrecan core protein steady state messenger RNA levels. Thus, the effects of PG on aggrecan production in RCJ cells appear to be regulated at the posttranscriptional level. Forskolin and (Bu)2cAMP mimicked the suppressive effects of PGE1 on [3H]TdR incorporation, as well as the stimulatory effect of PGE1 on aggrecan synthesis. In addition, the phorbol ester 12-O-tetradecanoyl phorbol acetate mimicked PGF2 alpha stimulation of [3H]TdR incorporation and aggrecan synthesis, and the effects of PGE2 alpha on these processes were blocked by protein kinase C inhibitors. Therefore, it appears that in mammalian chondrocytes, PGE1 primarily activates the cAMP-protein kinase A second messenger system, PGE2 alpha affects primarily the Ca2(+)-protein kinase C system, and PGE2 activates both pathways. Moreover, PG posttranscriptional regulation of aggrecan synthesis in chondrocytes involves both the cAMP-protein kinase A and Ca2(+)-protein kinase C second messenger systems.

Age-related changes in osteogenic stem cells in mice.

Osteoblasts arise from partially differentiated osteogenic progenitor cells (OPCs) which in turn arise from undifferentiated marrow stromal mesenchymal stem cells (MSCs). It has been postulated that age-related defects in osteoblast number and function may be due to quantitative and qualitative stem cell defects. To examine this possibility, we compared osteogenic stem cell number and in vitro function in marrow cells from 4-month-old and 24-month-old male BALB/c mice. Histologic studies demonstrated that these mice undergo age-related bone loss resembling that seen in humans. In primary MSC cultures grown in media supplemented with 10 nM dexamethasone, cultures from older animals yielded an average of 41% fewer OPC colonies per given number of marrow cells plated (p < 0.001). This implies that for a given number of marrow cells there are fewer stem cells with osteogenic potential in older animals than there are in younger animals. The basal proliferative rate in cultures from older animals, as measured by 3H-thymidine uptake, was more than three times that observed in cultures from young animals (p < 0.005). However, the increase in proliferative response to serum stimulation was 10-fold in the younger cultures (p <0.001) and insignificant (p <0.4) in the older cultures. Colonies in both age groups became alkaline phosphatase positive at the same rate, and virtually all colonies were positive after 12 days of culture. Cultures from both age groups produced abundant type I collagen. These studies suggest that defects in the number and proliferative potential of MSCs may underlie age-related defects in osteoblast number and function.

Osteoporosis in steroid-dependent asthma.

BACKGROUND: Patients on prolonged corticosteroid therapy are at risk of developing osteoporosis. Some patients with severe asthma are difficult to wean off corticosteroids and are therefore at risk of developing bony complications due to steroids. OBJECTIVE: The purpose of this study was to examine the relationship of cumulative steroid dosage and duration of therapy with osteoporosis. METHODS: We obtained bone mineral density studies using dual photon absorptiometry, and radiographs of the lumbar spine of 16 steroid-dependent patients with asthma. Patients with conditions affecting bone metabolism were excluded. RESULTS: We studied 16 male steroid-dependent patients with asthma who received 4 to 41 grams equivalent dose of prednisone over a period of 1 to 15 years. The overall prevalence rate for abnormal age-matched bone mineral density was 50%. Abnormal bone mineral density was more commonly noted in the lumbar spine (38%) than in the femoral neck (19%). The lowest dose of corticosteroid associated with a decrease in bone mineral density was a cumulative steroid dose of 5.6 equivalent grams-prednisone. CONCLUSION: Prolonged corticosteroid therapy can cause significant osteoporosis among male patients with steroid-dependent asthma. Bone loss due to corticosteroid therapy occurs at different rates at different bony sites.

The effect of a weighted vest on perceived health status and bone density in older persons.

To assess the effect of an exercise intervention using a weighted vest on perceived health status and bone density in older persons, we enrolled 36 seniors in a randomized controlled trial. The vest-use group met weekly for 1 h for a low level exercise class. They wore a weighted vest during the class and as tolerated at home. The discussion controls met for 1 h weekly. At baseline and follow-up (20 weeks), subjects completed a questionnaire that included the 20 item MOS Short-Form Health Survey, Multidimensional Health Locus of Control Scale, and Philadelphia Geriatric Center Morale Scale, and bone density was measured by dual energy X-ray absorptiometry. Subjects also completed daily activity diaries. Subjects in the vest group reported a statistically significant decrease in bodily pain, improved physical functioning, and increased internal health locus of control. Bone density increased by 1% in the vest group and decreased by 0.6% in the controls (p = 0.12). We conclude that our exercise intervention had a positive effect on some measures of perceived health in older persons.

Interrelationship between parathyroid hormone and insulin: effects on DNA synthesis in UMR-106-01 cells.

UMR-106-01 osteoblast-like cells respond to high concentrations of parathyroid hormone (PTH) in vitro by decreasing thymidine incorporation, a marker of DNA synthesis and cell proliferation. This response is different from in vivo conditions, such as primary and secondary hyperparathyroidism, in which high PTH levels are associated with an increased number of osteoblasts. When the response of UMR-106-01 cells to PTH is evaluated in vitro, however, these cells are exposed to only a single hormone. The present study was designed to evaluate the combined effects of two hormones, PTH and insulin, on the DNA synthesis of UMR-106-01 cells. PTH is known to decrease and insulin to increase thymidine incorporation by UMR-106-01 cells. To examine the interaction of these hormones, acute studies, defined as a 24 h exposure to hormone, and chronic studies, defined as a 7 day exposure to hormone, were performed. Both acute and chronic exposure to 10(-9) M PTH decreased thymidine incorporation by UMR-106-01 cells, with suppression ranging from 27 to 81% (P < 0.05). Both acute and chronic exposure to 10(-8) M insulin (INS) increased thymidine incorporation by UMR-106-01 cells; this ranged from 26 to 58% (P < 0.05). However, chronic exposure to 10(-9) M PTH followed by an acute exposure to 10(-8) M INS resulted in a 710% increase in thymidine incorporation (P < 0.01). Reversing the sequence by chronically exposing UMR-106-01 cells to 10(-8) M INS followed by acute exposure to 10(-9) M PTH resulted in a 53% decrease in thymidine incorporation (P < 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

The effects of prostaglandin E2, parathyroid hormone, and epidermal growth factor on mitogenesis, signaling, and primary response genes in UMR 106-01 osteoblast-like cells.

Prostaglandin E2 (PGE2), PTH, and epidermal growth factor (EGF) are potent regulators of osteoblast proliferation. In UMR 106-01 rat osteosarcoma cells with osteoblast-like features, PGE2 and PTH inhibit, while EGF stimulates, mitogenesis. Both PGE2 and PTH increase intracellular cAMP levels, cytosolic calcium, and inositol phosphate turnover. In a variety of cell types, EGF mediates its effects in part via activation of receptor protein-tyrosine kinase and other protein kinases, such as protein kinase-C. The nuclear mechanisms of PGE2, PTH, and EGF regulation of osteoblast proliferation are unknown. Accordingly, we have examined the effects of these agents on mitogenesis, second messenger generation, and primary response genes, which may link second messenger activation to subsequent alterations in gene expression. Northern blot analysis of mRNA from UMR 106-01 cells treated for 3 h with 2 microM PGE2, 10 nM PTH, or 10 ng/ml EGF in the presence of cycloheximide demonstrated that all three agents induced the expression of c-fos and c-jun mRNA. In contrast, only EGF stimulated cellular proliferation and induced Egr-1 mRNA. Also, unlike PGE2 and PTH, EGF did not increase intracellular cAMP levels. c-fos mRNA was induced by treatment with 50 ng/ml tetradecanoyl phorbol acetate or by 40 ng/ml forskolin, while induction of Egr-1 mRNA was stimulated by treatment with tetradecanoyl phorbol acetate, but not forskolin. Thus, EGF signal transduction differs from that of PGE2 and PTH in UMR 106-01 osteoblast-like cells, in that EGF does not stimulate the protein kinase-A second messenger system, but causes activation of Egr-1, a primary response gene that may play a role in the mitogenic effect of EGF.

Growth-associated glycosylation of transferrin secreted by HepG2 cells.

Growth-associated alterations in the oligosaccharide structures of transferrin secreted by HepG2 cells were examined by concanavalin A-crossed affinoimmuno-electrophoresis. Slowly dividing, confluent cultures produced transferrin with a approximately 4.5-fold greater proportion of biantennary complex-type oligosaccharides than rapidly dividing, subconfluent cultures. The activity of N-acetylglucosaminyltransferase V (GlcNAc-T V) and galactosyltransferase were measured in cell extracts from subconfluent and confluent cultures. While the activity of galactosyltransferase remained relatively constant, the activity of GlcNAc-T V was approximately 3.2-fold lower in confluent cultures than in subconfluent cultures. These results suggest that the growth-associated alteration in the oligosaccharides of transferrin is due, at least in part, to the regulation of GlcNAc-T V activity.

Effects of calcitonin on 3',5'-cyclic adenosine monophosphate and calcium second messenger generation and osteoblast function in UMR 106-06 osteoblast-like cells.

The UMR 106-06 rat osteosarcoma osteoblast-like cell line possesses calcitonin (CT) receptors in addition to expressing PTH receptors and a highly osteoblast-like phenotype, and may represent an intermediate developmental stage between early osteoblast precursors and mature osteoblasts. Therefore, we examined the effects of CT and PTH on second messenger generation and osteoblastic function in these cells. In UMR-106-06 cells, 10-1000 nM CT produced a dose-dependent stimulation of intracellular free calcium concentration ([Ca2+]i), which reached a plateau between 2-3 min. This stimulatory effect was abolished in the absence of extracellular Ca2+ ([Ca2+]o) and was mimicked by forskolin and (Bu)2cAMP. One hundred nanomolar CT also produced a slight but significant increase in inositol triphosphate production (13%, P less than 0.05) but did not produce a rapid, transient increase in [Ca2+]i. In contrast, PTH produced a rapid, transient increase in [Ca2+]i, which reached a maximum within 30 sec. This stimulatory effect of PTH on [Ca2+]i signal was dose-dependent and accompanied by a parallel stimulation of inositol triphosphate production. PTH, forskolin, and (Bu)2cAMP all produced a marked dose-related suppression of both DNA and collagen synthesis, which paralleled their stimulatory effects on intracellular cAMP levels. In marked contrast, CT only minimally reduced DNA and collagen synthesis despite producing comparable increases in intracellular cAMP. One hundred nanomolar CT also stimulated alkaline phosphatase specific activity by 33% (P less than 0.05). Thus, CT stimulates cAMP, [Ca2+]i, and inositol phosphate second messengers in UMR 106-06 cells. However, in contrast to other agents which elevate intracellular cAMP levels, CT does not suppress DNA synthesis. These results suggest that the linkage of CT receptor second messengers to effects on cell function differ from those of PTH and/or that CT may produce additional second messenger(s) which antagonize the antiproliferative effect of increased cAMP levels in UMR-106-06 cells.

Removal of the artifactual peak associated with concanavalin A crossed affinoimmunoelectrophoresis of human serum transferrin.

During the first dimension of crossed affinoimmunoelectrophoresis (CAIE) with the lectin concanavalin A (Con A), an immobile glycoprotein-Con A affinoprecipitate may form near the application well and, subsequently, produce an artifactual peak in the second-dimension gel. In this study, we examined the effects of sample glycoprotein concentration and gel Con A concentration on the magnitude of the transferrin artifactual peak present in the analysis of human serum. In addition, we examined the potential for reducing or eliminating the artifact by including a competitive inhibitor of glycoprotein-lectin interaction, alpha-methylmannoside (alpha MM), in the application well. We demonstrate that the artifact can be eliminated through an appropriate choice of glycoprotein, Con A, and alpha MM concentrations. This approach should be applicable for diagnosing and eliminating the artifact in the Con A CAIE analyses of other glycoproteins.

Insulin acutely suppresses parathyroid hormone second messenger generation in UMR-106-01 osteoblast-like cells: differential effects on phospholipase C and adenylate cyclase activation.

Insulin modifies the effects of PTH on osteoblast-like cells. However, the basis for this effect is unknown. In bone and kidney cells, the effects of PTH on cellular function are mediated by second messengers generated through both the phospholipase C and adenylate cyclase systems. Therefore, we examined the effects of insulin on PTH second messenger generation in UMR-106-01 rat osteoblastic osteosarcoma cells. PTH produced a rapid, transient increase in intracellular free calcium concentration ([Ca2+]i) which was maximal at 30 sec and was only minimally reduced in the absence of extracellular calcium. Inositol-triphosphate (IP3) production was increased in parallel. PTH stimulation of [Ca2+]i was concentration-dependent from 0.5-1,000 nM, with half-maximal stimulation at approximately 50 nM PTH. A 30-sec exposure to 50 nM PTH produced 32% and 23% increases in IP1 and IP3 production, respectively (both P less than 0.05). Although insulin alone did not significantly alter basal [Ca2+]i, a 1-min exposure to 1-100 nM insulin produced a concentration-dependent suppression of the PTH-stimulated transient increase in [Ca2+]i and IP3 generation. 100 nM insulin decreased 50 nM PTH stimulation of [Ca2+]i and IP3 levels by 84% (P less than 0.02) and 80% (P less than 0.001), respectively. Preexposure to insulin also decreased PTH stimulation of intracellular cAMP levels, but to a lesser degree. A 1-min exposure to 100 nM insulin produced a 32% (P less than 0.01) decrease in PTH-stimulated cAMP generation, but lower insulin concentrations were without significant effects. These results demonstrate that in UMR-106-01 cells, insulin suppresses PTH stimulation of second messengers generated through both the phospholipase C and adenylate cyclase systems, but has a more marked effect on the former.

The use of the Karnofsky Performance Scale in determining outcomes and risk in geriatric outpatients.

The Karnofsky Performance Scale (KPS) was evaluated in a geriatric outpatient population with regard to three issues: its strength of association with widely used and validated geriatric instruments; its ability to predict patient outcomes; and its ability to serve as an identifier of high-risk patients. The 134-patient sample was given a comprehensive geriatric assessment which included the KPS, the Activities of Daily Living (ADL) scale, the Instrumental Activities of Daily Living (IADL) scale, and other psychosocial and sensory tests. The KPS, ADL, and IADL were significantly correlated with each other, and the KPS showed the strongest associations with other functional measures. The KPS was also highly predictive of outcomes, performing better or equally well as the ADL and IADL. The KPS designation of high- and low-risk groups resulted in statistically significant score differences between groups in all but one assessment area, demonstrating better ability to discriminate than either the ADL or IADL. Thus, the KPS was shown to serve as an effective proxy score for a patient's health and functional status. It also was a significant predictor of hospitalizations, survival time, community residence, and institutionalization. Finally, the KPS was shown to adequately distinguish risk groups to aid in the targeting of services to ambulatory geriatric patients.

Effects of interleukin-6 on cellular function in UMR-106-01 osteoblastlike cells.

High levels of interleukin-6 (IL-6) have been detected in synovial fluid from patients with inflammatory arthropathies associated with local bone resorption, suggesting a role for IL-6 as a local regulator of bone resorption and remodeling. In the present study we examined the effects of IL-6 on [3H]thymidine ([3H]TdR) incorporation, collagen synthesis, and alkaline phosphatase activity in UMR-106-01 rat osteoblastic osteosarcoma cells. IL-6 stimulated a dose-dependent increase in [3H]TdR incorporation that was maximal at 1000 U/ml (-147% of basal, p less than 0.005) in osteoblastlike cells that were in a logarithmic phase of growth. The increase in [3H]TdR incorporation was maximal between 12 and 24 h and was neutralized by pretreatment with the polyclonal rabbit antibody to IL-6. IL-6 also increased cell number and the secretion of prostaglandin E2 in UMR-106-01 cells in logarithmic growth phase. The stimulation of [3H]TdR incorporation and release of PGE2 into the culture medium by IL-6 was inhibited by indomethacin. A 24 h exposure of the osteoblastlike cells to 1000 U/ml of IL-6 reduced [3H]proline incorporation into collagenase-digestible (CDP) protein to 73% of control values (p less than 0.01). Noncollagen protein (NCP) synthesis was inhibited to 80% of control values (p less than 0.01) by 1000 U/ml of IL-6. The inhibitory effect was relatively greater on CDP than on NCP and consequently resulted in a decrease in the percentage of collagen synthesis. Alkaline phosphatase activity was not altered in these cells after a 24 h exposure to 1-1000 U/ml of IL-6.(ABSTRACT TRUNCATED AT 250 WORDS)

Cellular immunosenescence: an overview.

Recent studies on space flights suggest that certain T cell immunologic activities are vulnerable to microgravitation. It would be desirable to know the extent to which these changes can be prevented or reversed. Since the changes observed are analogous to the effects of aging on immunity, a brief overview is presented of our current knowledge of age-related changes in immune cells and of the various interventional methods which have been used successfully in preventing the decline with age and in elevating the levels of immune functions of old individuals.

Effects of nicotine on cellular function in UMR 106-01 osteoblast-like cells.

We examined the effect of nicotine on cellular proliferation, as measured by [3H]thymidine (TdR) incorporation and cell count, and on alkaline phosphatase activity in UMR 106-01 rat osteoblastic osteosarcoma cells. The cells were cultured with varying concentrations of nicotine in serum-free medium for 2 to 72 hours. Nicotine produced a dose-dependent suppression of TdR incorporation, with maximum suppression seen at 10 mM (7% of control); the EC50 for suppression of TdR incorporation was 10 microM. 1 microM nicotine decreased cell number by 20% to 30%. The time course of the effect of 100 microM nicotine on DNA synthesis was measured by TdR incorporation. TdR uptake was measured at 2, 4, 6, 24, 48, and 72 hours. After the addition of nicotine, the following biphasic response in TdR incorporation was observed: a 15% decrease at 2 hours, recovery to near control value at 6 hours, a 27% decrease by 24 hours, and a maximum decrease of 88% by 48 hours. Over a dose range of 1 nM to 10 mM, nicotine produced a dose-dependent increase in alkaline phosphatase activity with maximum stimulation seen at 1 microM (189% of control). We conclude that nicotine suppresses cellular proliferation and stimulates alkaline phosphatase activity in UMR 106-01 osteoblast-like cells. These results may be of significance in the development of osteoporosis and alveolar bone loss associated with the use of tobacco.

Protein kinase C activity in UMR-106-01 cells: effects of parathyroid hormone and insulin.

The calcium and phospholipid-dependent protein kinase C (PKC) system appears to play an important role in mediating hormonal effects in various tissues including bone. Accordingly, we characterized PKC activity in the UMR-106-01 rat osteosarcoma osteoblastlike cell line and examined its hormonal regulation. UMR-106-01 cells were found to possess a classic, phorbol ester-activated PKC system, which was highly calcium and phospholipid dependent. A 30 s exposure to 10 nM bovine parathyroid hormone (PTH) (1-34) increased cytosolic and membrane-bound PKC activity by 12 and 157%, respectively, resulting in a 2.2-fold increase in the membrane-bound to cytosolic (MB/C) activity ratio (all p less than 0.01). The MB/C activity ratio was highest at 20 min, exhibiting a 2.8-fold increase over the control values (p less than 0.01). In contrast, 10 nM insulin increased cytosolic PKC activity but decreased membrane-bound activity, resulting in a 61% decrease in the MB/C activity ratio at 20 min (p less than 0.02). Moreover, insulin reduced PTH stimulation of the PKC activity ratio by 42 and 62% at 30 s and 20 min, respectively (p less than 0.02). Thus, PTH and insulin have opposing effects on the PKC activity ratio in UMR-106-01 cells.