Human mast cells induce osteoclastogenesis through cell surface RANKL.

OBJECTIVES: We employed the co-culture of CD34+ stem cell-derived human mast cells (HMC) and human monocyte-derived osteoclast precursors to evaluate if mast cells contribute to the pathogenesis of osteoporosis through regulation of osteoclast proliferation and activation. METHODS: Mature HMC and osteoclast precursors were cultured from monocytes isolated from human buffy coat. The osteoclast precursors were incubated with HMC or receptor activator of nuclear factor kappa-B ligand (RANKL) for a week prior to determination of osteoclast maturation through characterization by their morphology and tartrate resistant acid phosphatase (TRAP) expression. The bone absorption activity was determined by pit formation on osteo-assay plate. RESULTS: Mature osteoclasts were identified following co-culture of osteoclast precursors with HMC for one week in the absence of RANKL and they were capable of bone resorption. These actions of HMC on osteoclasts were not affected by mast cell activators such anti-IgE or substance P but could be reversed by osteoprotegerin (OPG) in the co-culture system suggesting the involvement of RANKL. The expression of RANKL on the cell surface of HMC was confirmed by flow cytometry and the density was not affected by activation of HMC. CONCLUSION: Our study provided direct evidence confirming the initiation of osteoclast proliferation and activation by mast cells through cell surface RANKL suggesting that mast cells may contribute to bone destruction in pathological conditions such as osteoporosis.

Novel six-week protocol for generating functional human connective tissue-type (MCTC) mast cells from buffy coats.

OBJECTIVE: The aim of this study was to develop a novel protocol for generating large populations of fully mature and functional human mast cells (HMC) from CD34+ hematopoietic stem cells which require less culturing time than previously reported methods. METHODS: CD34+ cells isolated from fresh human buffy coats were sequentially cultured with different combinations of SCF, IL-6, IL-3, IL-9 and IL-4 under selected culturing conditions and time periods. Cells were then harvested for immunohistochemical characterization of morphological phenotypes and were functionally characterized by assessing their responses to IgE-dependent and -independent stimuli by measuring the release of inflammatory mediators and cytokines. Moreover, the pharmacological profiles of several classes of anti-inflammatory drugs in inhibiting the activation of these HMC were also characterized. RESULTS: We have developed a novel protocol that can generate large homogenous populations of mature and functional HMC in 6 weeks. These cells expressed both tryptase and chymase and were activated by anti-IgE, cationic peptides and calcium ionophores. Moreover, IgE-dependent activation of these cells was significantly inhibited by anti-inflammatory drugs. The morphological and functional characteristics of these mast cells resembled those of MCTC type or connective tissue-type HMC. DISCUSSION: Our protocol represents a novel time-saving and economical approach for generating large numbers of primary HMC for functional studies of mast cell biology and for profiling novel anti-inflammatory therapeutic agents with mast cell-inhibitory properties in humans.

Suppression of mast cell activity contributes to the osteoprotective effect of an herbal formula containing Herba Epimedii, Fructus Ligustri Lucidi and Fructus Psoraleae.

OBJECTIVES: Mast cells are believed to contribute to the pathogenesis of osteoporosis as their number is increased in osteoporotic bones. Herba Epimedii, Fructus Ligustri Lucidi and Fructus Psoraleae are three Chinese herbs traditionally for tonifying the 'kidney system' and a herbal formula (ELP) containing the respective herbs at the weight ratio of 5 : 4 : 1 was shown to prevent osteoporosis. This study evaluated if suppression of mast cell accumulation and activity contribute to the anti-osteoporotic action of ELP. METHODS: The herbs were boiled under reflux to produce the aqueous extract that was further concentrated under reduced pressure and lyophilized. An in-vivo rat osteoporosis model using hind limb unloading was employed for studying the accumulation of mast cells. The human mast cell line, LAD2, was employed to evaluate the mast cell modulating action of ELP. KEY FINDINGS: Mast cell number in the tibiae of hind limb unloaded rats increased significantly during the course of osteoporosis. ELP treatment (10 g/kg/day) prevented both osteoporosis and mast cell accumulation in these rats. Furthermore, ELP significantly inhibited histamine and tumour necrosis factor-α release from LAD2 cells. CONCLUSION: Mast cells contributed to hormone independent osteoporosis. The suppression of mast cell accumulation and activation may contribute to the anti-osteoporotic action of ELP.