Interleukin-6 messenger RNA expression and interleukin-6 protein secretion in cells isolated from normal human bone: regulation by interleukin-1.

Recent evidence suggests that cytokines, in addition to regulating hematopoiesis and immune functions, may be important paracrine regulators of bone turnover. Interleukin-1 (IL-1) and IL-6 are cytokines that are produced by and affect both hematopoietic and nonhematopoietic cell types. IL-1 stimulates bone resorption and inhibits osteoblast proliferation and collagen production. Previous reports that IL-6 was secreted in murine osteoblast and bone organ cultures in response to IL-1 and PTH suggested that IL-6 has paracrine effects on bone resorption or formation. To determine whether IL-6 has a paracrine function in human bone, IL-6 expression in cells isolated from normal human bone was investigated. IL-6 mRNA levels in untreated cultures were low and variable, and IL-6 secretion was undetectable. PTH had no effect on IL-6 mRNA levels or IL-6 secretion. IL-1 beta increased IL-6 mRNA levels, maximally 40-fold at 12 h. IL-1 beta increased IL-6 secretion to 0.13 nM, more than 80-fold that of untreated controls at 12 h. IL-1 beta also increased IL-1 beta mRNA levels, maximally 9-fold at 12 h, but did not increase cellular levels or secretion of IL-1 beta protein. Recombinant human IL-6 at 0.5-5 nM stimulated resorption in neonatal mouse calvarial organ cultures but had no effect on human bone-derived cell DNA synthesis or type I procollagen mRNA levels. The results suggest that IL-6 production by human osteoblasts may function to enhance osteolytic activity of IL-1 but does not affect proliferative and matrix biosynthetic aspects of bone formation that were tested. Because osteoblasts and bone marrow cells are in close proximity, IL-6 produced by osteoblasts may also function to amplify IL-1 stimulation of immune responses and hematopoiesis in bone marrow.

Osteoclast formation in bone marrow cultures from two inbred strains of mice with different bone densities.

For the purpose of identifying genes that affect bone volume, we previously identified two inbred mouse strains (C57BL/6J and C3H/HeJ) with large differences in femoral bone density and medullary cavity volume. The lower density and larger medullary cavity volume in C57BL/6J mice could result from either decreased formation or increased resorption or both. We recently reported evidence suggesting that bone formation was increased in vivo and that osteoblast progenitor cells are more numerous in the bone marrow of C3H/HeJ compared with C57BL/6J mice. In the present study, we determined whether osteoclast numbers in vivo and osteoclast formation from bone marrow cells in vitro might also differ between the two mouse strains. We have found that the number of osteoclasts on bone surfaces of distal humerus secondary spongiosa was 2-fold higher in 5.5-week-old C57BL/6J mice than in C3H/HeJ mice of the same age (p < 0.001). Bone marrow cells of C57BL/6J mice cocultured with Swiss/Webster mouse osteoblasts consistently produced more osteoclasts than did C3H/HeJ bone marrow cells at all ages tested from 3.5-14 weeks of age (p < 0.001). Osteoclast formation was also greater from spleen cells of 3.5-week-old C57BL/6J mice than C3H/HeJ mice. The distribution of nuclei per osteoclast and the 1, 25-dihydroxyvitamin D3 dose dependence of osteoclast production from bone marrow cells were similar. Osteoclasts that developed from both C57BL/6J and C3H/HeJ marrow cells formed pits in dentin slices. Cultures from C57BL/6J marrow cells formed 2.5-fold more pits than cultures from C3H/HeJ marrow cells (p < 0.02). We compared the abilities of C57BL/6J and C3H/HeJ osteoblasts to support osteoclast formation. When bone marrow cells from either C57BL/6J or C3H/HeJ mice were cocultured with osteoblasts from either C57BL/6J or C3H/HeJ newborn calvaria, the strain from which osteoblasts were derived did not affect the number of osteoclasts formed from marrow cells of either strain. Together, these observations suggest that genes affecting the bone marrow osteoclast precursor population may contribute to the relative differences in bone density that occur between C3H/HeJ and C57BL/6J mouse strains.

Multiple osteocalcin fragments in human urine and serum as detected by a midmolecule osteocalcin radioimmunoassay.

Reliable markers of bone formation are essential to the investigation of metabolic bone disorders. In this regard, evidence indicates that circulating levels of human osteocalcin (OC) correlate with the skeletal isoenzyme of alkaline phosphatase and can be used as an index of bone formation. A disadvantage of using serum OC as a marker of formation is its diurnal variation. To address this problem we carried out our studies to determine the usefulness of urine in the assessment of bone turnover. Using a midmolecule specific human OC RIA, we were able to detect OC in urine of normal adults (42 mugeq/g creatinine), normal children (849 mu/geq/g creatinine), and Paget's disease patients (613 mugeq/g creatinine). Immunoreactive fragments of OC in human urine and human serum were separated by high pressure liquid chromatography. Multiple fragments were found in normal adult urine that were not detected in normal adult serum. Uremic and Paget's disease sera contain several immunoreactive forms of OC, other than the intact molecule, not found in normal adult serum. Additionally, both Paget's disease sera and urine contained a specific peak of immunoreactive material, eluting at 25% acetonitrile, that was not found in any other serum or urine tested. Urinary OC (uOC) correlated with both skeletal alkaline phosphatase (r = 0.91) and serum OC (r = 0.83), indices of skeletal formation. While uOC has a diurnal variation similar to that of serum OC, determinations of 24-h uOC give integrated values of daily bone turnover rates. Z-Score analysis indicates that uOC (z = 14.04) is better able to distinguish between normal children with high bone turnover and normal adults than either skeletal alkaline phosphatase (z = 8.87) or serum OC (z = 9.01).

Calcium deficiency in fluoride-treated osteoporotic patients despite calcium supplementation.

To test the hypothesis that the osteogenic response to fluoride can increase the skeletal requirement for calcium, resulting in a general state of calcium deficiency and secondary hyperparathyroidism, we assessed calcium deficiency, spinal bone density, by quantitative computed tomography, and serum PTH in three groups of osteoporotic subjects. Two of the three groups had been treated with fluoride and calcium (at least 1500 mg/day) for 32 +/- 19 months. Group 1 consisted of 16 fluoride-treated subjects who had shown rapid increases in spinal bone density (+ 3.8 +/- 2.6 mg/cm2 month), group II consisted of 10 fluoride-treated subjects who had shown decreases or only slow increases in spinal bone density (-0.05 +/- 0.6 mg/cm3 month), and group III consisted of 10 age-matched untreated osteoporotic controls. Calcium deficiency was assessed by measurement of calcium retention after calcium infusion. The results of our studies showed that 1) 94% of the subjects in Group I were calcium deficient compared with only 30% in groups II and III (P < 0.01 for each); 2) the subjects in group I retained more calcium (79%) than the subjects in group II (60%, P < 0.001) or the subjects in group III (64%, P < 0.005); 3) calcium retention was proportional to serum PTH (r = 0.37, n = 36, P < 0.03); and 4) calcium retention was proportional to the (previous) fluoride-dependent increase in quantitative computed tomography spinal bone density (in groups I and II, r = 0.48, n = 26, P < 0.02). To test the hypothesis that the calcium deficiency and the secondary hyperparathyroidism that were associated with the positive response to fluoride would respond to concomitant calcitriol treatment, a subgroup of 7 calcium-deficient subjects were selected from group I and treated with calcitriol (plus fluoride and calcium) for an average of 7 months. The calcitriol therapy reduced the calcium deficit in all 7 subjects, decreasing calcium retention from 80% to 62% (P < 0.02), and decreasing PTH from 50 to 28 pg/mL (P < 0.02). Together, these data indicate that fluoride-treated osteoporotic subjects may develop calcium deficiency in proportion to the effect of fluoride to increase bone formation, and this calcium deficit is responsive to calcitriol therapy.

Alkaline phosphatase levels and osteoprogenitor cell numbers suggest bone formation may contribute to peak bone density differences between two inbred strains of mice.

Previous studies have shown that C3H/HeJ (C3H) mice have higher peak bone density than C57BL/6J (B6) mice, at least in part because of differences in rates of bone resorption. The current studies were intended to examine the alternative, additional hypothesis that the greater bone density in C3H mice might also be a consequence of increased bone formation. To that end, we measured two presumptive, indirect indices of bone formation and osteoblast number in these inbred strains of mice: alkaline phosphatase (ALP) activity in serum, bones, and bone cells; and the number of ALP-positive colony-forming units (CFU) in bone marrow stromal cell cultures. We found that C3H mice had higher serum levels of ALP activity than B6 mice at 6 (118 vs. 100 U/L, p < 0.03) and 32 weeks of age (22.2 vs. 17.2 U/L, p < 0.001). Tibiae from C3H mice also contained higher levels of ALP activity than tibiae from B6 mice at 6 (417 vs. 254 mU/mg protein, p < 0.02) and 14 weeks of age (132 vs. 79 mU/mg protein, p < 0.001), as did monolayer cultures of bone-derived cells from explants of 7.5-week-old C3H calvariae and femora (8.2 times more, p < 0.02, and 4.6 times more, p < 0.001, respectively). Monolayer cell cultures prepared by collagenase digestion of calvariae from newborn and 6-week-old mice also showed similar strain-dependent differences in ALP-specific activity (p < 0.001 for each). Our studies also showed more ALP-positive CFU in bone marrow stromal cell cultures from 8-week-old C3H mice, compared with B6 mice (72.3 vs. 26.1 ALP-positive CFU/culture dish, p < 0.001). A similar result was seen for ALP-positive CFU production at 6 and 14 weeks of age, and the difference was greatest for the CFU that contained the greatest numbers of ALP-positive cells. Because skeletal ALP activity is a product of osteoblasts and has been shown to correlate with rates of bone formation, and because the number of ALP-positive CFU is believed to reflect the number of osteoprogenitor cells, the current data are consistent with the general hypothesis that bone formation may be greater in C3H than B6 mice because of a difference in osteoblast number. Our data further suggest that peak bone density may be greater in C3H mice than B6 mice due to a combination of decreased bone resorption and increased bone formation.

Circulating and skeletal insulin-like growth factor-I (IGF-I) concentrations in two inbred strains of mice with different bone mineral densities.

Recent work has demonstrated differences in femoral bone mineral density between two common inbred strains of mice, C3H/HeJ (C3H) and C57BL/6J (B6), across a wide age range. To investigate one possible mechanism that could affect acquisition and maintenance of bone mass in mice, we studied circulatory and skeletal insulin-like growth factor-I (IGF-I) and femoral bone mineral density (F-BMD) by pQCT in C3H and B6 progenitor strains, as well as serum IGF-I obtained from matings between these two strains and mice bred from subsequent F1 intercrosses (F2). Serum IGF-I measured by radioimmunoassay was more than 35% higher in virgin progenitor C3H than virgin B6 at 1, 4, 8, and 10 months of age, and in 8-month-old C3H compared with B6 retired breeders (p < 0.001). In the progenitors, there was also a strong correlation between serum IGF-I and serum alkaline phosphatase (r = 0.51, p = 0.001). In the 4 month F1 females IGF-I levels and F-BMD were intermediate between C3H and B6 progenitors. In contrast, groups of F2 mice with the highest or lowest BMD also had the highest or lowest serum IGF-I (p = 0.0001). IGF-I accounted for > 35% of the variance in F-BMD among the F2 mice. Conditioned media from newborn C3H calvarial cultures had higher concentrations of IGF-I than media from B6 cultures, and cell layer extracts from C3H calvariae exhibited greater alkaline phosphatase activity than cultures from B6 calvarial cells (p < 0.0001). The skeletal content of IGF-I in C3H tibiae, femorae, and calvariae (6-14 weeks of age) was also significantly higher than IGF-I content in the same bones of the B6 mice (p < 0.05). These data suggest that a possible mechanism for the difference in acquisition and maintenance of bone mass between these two inbred strains is related to systemic and skeletal IGF-I synthesis.

Development of a new radioimmunoassay for human osteocalcin: evidence for a midmolecule epitope.

Osteocalcin is a vitamin K-dependent bone-specific protein that can be found circulating in the serum. The circulating levels of osteocalcin have been shown to be an index of bone turnover. Existing radioimmunoassays for osteocalcin have been shown to be specific for C-terminal epitopes, a region that is identical in the human and bovine osteocalcin. There are, however, five amino acids different in the N-terminal region of the molecule. We describe here an immunoassay for a midmolecule epitope of osteocalcin using osteocalcin purified from human femoral head bone powder. Antibody specificity was determined using tryptic digests and a synthetic fragment of human osteocalcin. This assay has only a partial crossreactivity with bovine osteocalcin. This is the first report of an assay against a midmolecular epitope of osteocalcin involving a region in which the human and bovine osteocalcins differ. Osteocalcin levels determined by this assay have a significant correlation with both the total serum alkaline phosphatase and the serum skeletal alkaline phosphatase levels in normal adult human serum and, to a greater degree, in sera of patients with conditions associated with increased bone turnover (Paget's disease, hyperparathyroidism, and newborn sera). These correlations are greater than those previously reported for C-terminal assays, suggesting the possibility that different regions of the molecule may elicit different information concerning bone turnover.

Incidence of acetylcholinesterase in the sarcoplasm of human and chicken muscles.

Fifty-nine biopsies of human muscle, 53 of them abnormal, 6 normal, were studied for the histochemical localization of acetylcholinesterase (AChE) using frozen sections and light microscopy. In addition to AChE which was found at the myoneural and myotendon junction, specific staining was found around the periphery of many fibers from normal and abnormal muscles. Moreover, AChE activity was found to be high in the sarcoplasm of more than 10% of the fibers from 28 biopsies of abnormal muscle including cases of hemiplegia, spinal cord injury, denervation and neuropathy, infantile spinal muscle atrophy, Duchenne, limb-girdle and facioscapulohumeral dystrophies, Schwartz-Jampel syndrome and a myasthenic syndrome. Of the muscles from experimental animals examined, only the Rhesus monkey exhibited AChE around the periphery of the fibers, and only the dystrophic chicken and not the dystrophic mouse or hamster, showed extensive sarcoplasmic AChE. Histograms of muscle fiber diameters indicated that AChE in the sarcoplasm was associated with fibers of all sizes, depending on the nature of the disorder examined. Fibers containing AChE were smaller than unstained fibers in dystrophic chicken muscle. The results suggest that in the human, sarcoplasmic AChE is reversibly repressed during muscle maturation and that its mode of regulation by motor neurons is similar to that found in the chicken.

Human skeletal growth factor stimulates collagen synthesis and inhibits proliferation in a clonal osteoblast cell line (MC3T3-E1).

Human skeletal growth factor (hSGF), an 11-kD polypeptide purified from human bone, has been proposed to be a local regulator of bone formation. To investigate the underlying cellular mechanisms in an in vitro model system, we examined the effects of hSGF on proliferation and collagen synthesis in cells of the clonal osteoblast cell line MC3T3-E1. This line was isolated from newborn mouse calvarial cells and retains many characteristics of mature osteoblasts (Sudo, H., et al., (1984) J. Cell Biol. 96:191). A 14-hr treatment with hSGF increased noncollagenous protein synthesis to 215% of unstimulated controls and increased collagen synthesis to 630% of controls as determined by [3H]proline incorporation and high-pressure liquid chromatographic separation of [3H]proline and [3H]hydroxyproline in acid hydrolysates of trichloroacetic acid-insoluble protein. HSGF did not increase cell number over a 48-hr period and caused a reversible inhibition of DNA synthesis. Half-maximal hSGF concentration for stimulation of [3H]proline incorporation and inhibition of [3H]thymidine incorporation was 100 ng/ml. HSGF also inhibited DNA synthesis in cells stimulated by serum. In contrast, hSGF stimulated both collagen synthesis and DNA synthesis in primary cultures of chick embryo bone cells, which may be developmentally less mature than MC3T3-E1 cells. The results suggest that hSGF directly stimulated mature osteoblast matrix synthetic activity and that hSGF has differential effects on proliferation of osteoblast progenitor cells and mature osteoblasts.

Incorporation of sodium fluoride into cortical bone does not impair the mechanical properties of the appendicular skeleton in rats.

Clinical studies on the use of sodium fluoride (NaF) in osteoporotic patients have demonstrated increased spinal bone mass without a reduction in vertebral fracture incidence, and a trend towards reduced appendicular bone mass with an increase in peripheral fracture incidence. As previous reports have suggested that NaF becomes incorporated into bone's crystal structure, possibly affecting bone strength, we sought to examine the relationship among bone fluoride content, bone mass, and skeletal fragility. Twenty-one-day-old female Sprague-Dawley rats were treated with four different doses of NaF. The tibiae were subjected to histomorphometric and biochemical analyses, and the femora were tested in torsion for the properties of strength, stiffness, energy storage capacity, and angular deformation. The results showed that over 50% of the skeleton in these rats was turned over in the presence of NaF. The four different doses resulted in a linear increase in bone F concentration and suggested excellent absorption and incorporation of this drug. No changes in histomorphometric indices of bone formation or turnover were found. Despite the large fraction of bone formed during NaF treatment, and the linear increase in bone fluoride content in relation to dose, there were no changes observed in any of the mechanical properties. These results suggest that, even extensive incorporation of fluoride into bone, in the absence of an effect on bone mass or remodeling, does not significantly alter its capacity to withstand mechanical loads.

Synthetic peptide-based immunoassay for amino-terminal propeptide of type I procollagen: application for evaluation of bone formation.

Serum biochemical markers are powerful tools for the evaluation of bone turnover. In this study, we developed a radioimmunoassay, using a synthetic peptide for the N-terminal fragment of human type I [alpha 1(I)] procollagen (N-PCP). A 14-amino acid peptide was synthesized from the amino terminus and used to generate antibodies in rabbits. The synthetic peptide was used as standard and tracer in the assay. Both native type I amino procollagen (PINP), which was purified from skin fibroblasts, and human serum displaced tracer binding in parallel with the synthetic peptide. The range for measurement of N-PCP in serum was 0.7 to 30 micrograms/L (0.21-9.18 nmol/L). In a sample of 17 normal adults and 13 children (ages 9-16 years) there was a strong correlation between serum N-PCP determined by this assay and both skeletal alkaline phosphatase isoenzyme and osteocalcin, markers of bone formation. Serum concentrations of N-PCP in a group of normal children were eightfold higher than concentrations in normal adults, with no overlap between the two groups. N-PCP also correlated with C-terminal type I procollagen determined with a commercially available kit (r = 0.92).

Acetylcholinesterase in abnormal human muscle.

Fifty human muscle biopsies were examined for histochemical localization of acetylcholinesterase (AChE) activity. Six normal muscle samples had AChE at the myoneural junctions and around the periphery of many fibers. The AChE within the sarcoplasm itself was found in only a few atrophied fibers. However, 21 of 44 biopsies of abnormal muscles had sarcoplasmic AChE in more than 10% of their fibers. Such cases included Duchenne, limb-girdle and facio-scapulo-humeral dystrophy, neurogenic and spinal muscle atrophy, spinal cord injury, peripheral nerve injury, Schwartz-Jampel syndrome and myasthenic syndrome. Sarcoplasmic AChE is found in embryo muscle and usually declines after birth. It appears after denervation in the chicken but not the rat and remains in muscles of chickens with an inherited muscular dystrophy. The results of the human muscle study support the idea that in the human, as in the chicken, interruption of a neurally-mediated regulation of AChE results in the reappearance of high AChE activity in the sarcoplasm of the muscle fibers.