Interleukin-6 enhances hypercalcemia and bone resorption mediated by parathyroid hormone-related protein in vivo.

Tumors frequently induce the multifunctional cytokine IL-6, which has been linked to several paraneoplastic syndromes, most notably cachexia. IL-6 stimulates osteoclast formation, causes mild hypercalcemia, and is produced by bone cells in vitro upon exposure to systemic hormones. Since IL-6 is produced together with parathyroid hormone-related protein (PTH-rP) in some patients with cancer, we tested the hypothesis that production of IL-6 potentiates the effects of PTH-rP on Ca2+ homeostasis and osteoclastic bone resorption and examined potential mechanisms for these interactions in vivo. Chinese hamster ovarian (CHO) cells stably transfected with cDNAs for IL-6 (CHO/IL-6) and PTH-rP sense (CHO/PTH-rP) or antisense (CHO/PTH-rP AS) were inoculated intramuscularly into nude mice. Experimental groups included CHO/IL-6 plus CHO/PTH-rP; CHO/IL-6 plus CHO/PTH-rP AS; CHO/IL-6 alone; and CHO/PTH-rP alone. Blood ionized Ca2+ was measured on days 0, 7, 10, 12, and 13. Three different developmental stages in the osteoclast lineage were examined at day 13: the early multipotential precursor, granulocyte macrophage colony-forming units (CFU-GM); more mature mononuclear osteoclast precursors, assessed by their capacity to form tartrate-resistant acid phosphatase-positive multinucleated cells in marrow cultures; and mature osteoclasts, assessed by histomorphometry. IL-6 increased CFU-GM but not bone resorption or Ca2+. In contrast, PTH-rP induced hypercalcemia and bone resorption and increased multinucleated osteoclasts and more mature precursors cells, but not CFU-GM. However, mice treated with both IL-6 and PTH-rP had very marked hypercalcemia and osteoclastosis as well as an increase in the number of both CFU-GM and mature osteoclast precursors. These data demonstrate that IL-6 enhances PTH-rP-mediated hypercalcemia and bone resorption, most likely by increasing the pool of early osteoclast precursors that in turn can differentiate to mature osteoclasts. We conclude that IL-6 stimulatory effects on osteoclast precursors may enhance the effects of other bone resorption factors that act at later stages in the osteoclast lineage.

Tumor necrosis factor enhances parathyroid hormone-related protein-induced hypercalcemia and bone resorption without inhibiting bone formation in vivo.

Humoral hypercalcemia of malignancy results from the effects of tumor-produced factors on bone, kidney, and intestine that disrupt normal calcium homeostasis. Although parathyroid hormone-related protein (PTHrP) is a major mediator of the syndrome, tumors also produce other hypercalcemic factors, such as tumor necrosis factor (TNF), which may modulate the effects of PTHrP. It has been postulated that TNF may counteract the stimulatory effects of PTHrP on bone formation. To examine the effects of TNF on PTHrP-induced changes in calcium and bone metabolism, a murine tumor model of hypercalcemia was used. Nude mice were inoculated with Chinese hamster ovarian (CHO) cells expressing human TNF (CHO/TNF) or nontransfected CHO cells (CHO/-) and further treated with injections of human PTHrP(1-34) or vehicle. The effects of TNF, PTHrP, and the combination of the two factors on blood ionized calcium, osteoclast recruitment, and bone histomorphometry were evaluated. Mice bearing CHO/TNF tumors that were injected with PTHrP had significantly higher calcium concentrations, increased committed osteoclast progenitors, and mature osteoclasts as well as enhanced bone resorption compared with mice bearing CHO/TNF tumors injected with vehicle or those bearing CHO/- tumors injected with PTHrP or vehicle. A 2-fold increase in new woven bone formed in the calvaria at sites of previous bone resorption was observed in CHO/TNF mice treated with PTHrP. Bone formation rates in the vertebrae were similar in both CHO/- and CHO/TNF mice treated with PTHrP. These data demonstrate that the hypercalcemic effects of PTHrP are enhanced by TNF and that this effect is due to the increased production of committed osteoclast precursors with a subsequent increase in osteoclastic bone resorption. Furthermore, PTHrP caused a coupled increase in osteoclastic bone resorption and new bone formation that was not inhibited by TNF. These findings highlight the complex interactions that may occur between tumor-produced factors on bone that result in malignancy-associated hypercalcemia and suggest that TNF may not be responsible for the decreased bone formation seen in some patients with this condition.

Bisphosphonates promote apoptosis in murine osteoclasts in vitro and in vivo.

Bisphosphonates inhibit bone resorption and are therapeutically effective in diseases of increased bone turnover, such as Paget's disease and hypercalcemia of malignancy. The mechanisms by which they act remain unclear. Proposed mechanisms include inhibition of osteoclast formation from precursors and inhibitory or toxic effect on mature osteoclasts. We have developed a new in vitro model to study osteoclast survival and in this paper present in vitro and in vivo evidence that may explain both the observed reduction in osteoclast numbers and in bone resorption by mature osteoclasts, namely that bisphosphonates induce programmed cell death (apoptosis). Three bisphosphonates (risedronate, pamidronate, and clodronate) caused a 4- to 24-fold increase in the proportion of osteoclasts showing the characteristic morphology of apoptosis in vitro. This observation was confirmed in vivo in normal mice, in mice with increased bone resorption, and in nude mice with osteolytic cancer metastases, with similar-fold increases to those observed in vitro. Of the three compounds, risedronate, the most potent inhibitor of bone resorption in vivo, was the strongest inducer of osteoclast apoptosis in vitro. Osteoclast apoptosis may therefore be a major mechanism whereby bisphosphonates reduce osteoclast numbers and activity, and induction of apoptosis could be a therapeutic goal for new antiosteoclast drugs.

Use of an in vivo model to determine the effects of interleukin-1 on cells at different stages in the osteoclast lineage.

In vitro model systems have been used extensively to study factors that affect osteoclast formation and to identify osteoclast precursors. However, in vitro systems do not examine the entire process of osteoclast differentiation simultaneously and lack accessory cells normally present in vivo. Additionally, the role that metabolism of the factor may play on its osteotropic activity in vivo is not addressed by these culture systems. Therefore, we have developed an in vivo model that permits us to examine simultaneously the effects of osteotropic factors on three distinct stages of osteoclast differentiation: (1) multipotent osteoclast precursors, the granulocyte-macrophage colony-forming unit (CFU-GM); (2) more differentiated marrow mononuclear osteoclast precursors; and (3) mature osteoclasts already present on bone surfaces. In the current study, we used interleukin-1 (IL-1) as a prototypic osteotropic factor to test the utility of this system to delineate the cellular mechanisms responsible for enhanced osteoclast activity stimulated by this cytokine. IL-1 induced hypercalcemia and enhanced the growth and differentiation of CFU-GM, increased the number of more committed mononuclear osteoclast precursors, and stimulated mature osteoclasts to resorb bone. These data demonstrate that this simple in vivo model permits the easy delineation of the stages of osteoclast development, in which osteotropic factors act to enhance bone turnover, and may be useful in understanding the mechanism of action of antiresorptive agents.

Effects of parathyroid hormone (PTH)-related protein and PTH on osteoclasts and osteoclast precursors in vivo.

Increased production of PTH-related protein (PTHrP) and PTH is frequently responsible for hypercalcemia and its associated morbidity. However, it is unclear whether these peptides produce identical effects on cells in the osteoclast lineage in vivo. To examine the effects of continuous in vivo exposure to these factors on both the osteoclast precursors and mature osteoclasts, we inoculated Chinese hamster ovarian cells expressing PTH-(1-84), PTHrP-(1-141), or nontransfected Chinese hamster ovarian cells into nude mice. The effects of these tumors on blood ionized calcium, plasma PTH and PTHrP concentrations, and osteoclast formation were then determined. PTH and PTHrP tumor-bearing mice became hypercalcemic (1.90 +/- 0.04 and 1.97 +/- 0.16 mmol/liter, respectively) compared with control mice (1.29 +/- 0.015 mmol/liter). After 4 days of hypercalcemia, mice were killed, and bone marrow cells were harvested to examine cells at three discrete stages of osteoclast development: multipotent osteoclast precursors, the granulocyte/macrophage colony-forming unit; more committed marrow mononuclear osteoclast precursors; and mature osteoclasts. Neither PTH nor PTHrP had an effect on granulocyte/macrophage colony-forming unit, but similarly increased the number of more committed mononuclear osteoclast progenitors as well as mature osteoclasts in the calvaria. No differences were detected between the effects of PTH and PTHrP on cells in the osteoclast lineage in vivo. Thus, PTH and PTHrP appear to affect only more differentiated cells in the osteoclast lineage, and the differences in osteoclastic bone resorption between primary hyperparathyroidism and humoral hypercalcemia of malignancy are probably due to mechanisms other than effects on osteoclast precursor cells in vivo.

Pattern of recovery of the hypothalamic-pituitary-thyroid axis following radioactive iodine therapy in patients with Graves' disease.

PURPOSE: To characterize the time course of recovery of the hypothalamic-pituitary-thyroid (HPT) axis by determining the frequency, onset, duration, and clinical attributes of the central hypothyroid phase following 131I therapy for Graves' disease and to examine whether the central hypothyroid phase is due to direct pituitary thyrotroph suppression or to hypothalamic thyrotropin-releasing hormone (TRH) deficiency. PATIENTS AND METHODS: Twenty-one hyperthyroid patients with Graves' disease evaluated at a university endocrine clinic and treated with radioactive iodine were prospectively studied. Serial thyroid function levels (serum thyroxine [T4], free thyroxine [free T4], triiodothyronine [T3], and thyroid-stimulating hormone [TSH]) were measured and TRH stimulation tests were performed at 2 to 4 week intervals for all subjects following 131I treatment. None of the patients was treated with thionamides after receiving 131I therapy. RESULTS: Nineteen (90%) of the patients with Graves' disease experienced a transient central hypothyroid phase defined as the presence of a suppressed or inappropriately normal TSH level despite a low free T4 level following 131I treatment. This phase occurred a mean of 62.8 +/- 5.1 days following 131I treatment, persisted for an average of 24.7 +/- 2.4 days, and was not predictive of eventual treatment outcome. All patients had concordantly low T4 and T3 levels during this period and exhibited a blunted TSH response to TRH compared to 29 euthyroid control subjects, suggesting primary feedback suppression at the level of the pituitary thyrotrophs. The suppressed thyrotrophs required a minimum of 2 weeks to recover once patients became hypothyroid. The length of preexisting hyperthyroidism, basal free T4 elevation, and administered dose of 131I failed to predict the duration of the central hypothyroid phase, although a higher dose of 131I was associated with an earlier onset of central hypothyroidism (r = -.51, P < 0.05). CONCLUSIONS: Clinicians should be aware of the delay in the recovery of the HPT axis that occurs in the majority of patients with Graves' disease treated with 131I and is manifested by a transient central hypothyroid phase. The blunted TSH response to TRH stimulation during this period suggests that suppression occurs primarily at the level of the pituitary thyrotrophs. The use of sensitive TSH measurements alone to monitor these patients during this period is not helpful and may be misleading.

Elevated thyroxine levels in a euthyroid patient. A search for the cause of euthyroid hyperthyroxinemia.

A clinically euthyroid man with a family history of hyperthyroidism presented for evaluation of an elevated thyroxine (T4) level and an increased free T4 index with a normal thyrotropin (TSH) level. Results of thyroid hormone-binding protein tests confirmed the diagnosis of familial dysalbuminemic hyperthyroxinemia. This disorder should be considered in patients who have a normal serum TSH level, despite an elevated total T4 concentration. Accurate diagnosis is essential to avoid inappropriate treatment. Affected family members also should be identified. No treatment is required, because patients remain euthyroid and maintain a normal free T4 level.