engleski

Bone phenotype of IL-7 transgenic mice

Interleukin 7 (IL-7, a product of stromal cells, is a cytokine that performs critical functions early lymphoid development of both T and B cells in mice, and of T cells in man. It was first discovered as a factor that promoted the growth of murine B cell precursors. Several transgenic mouse lines have been produced that express IL-7 under different promoters and develop various phenotypes, ranging from benign increase in T and B cells to lymphoproliferative disorders. Our goal is to investigate the bone phenotype in a transgenic mouse line in which the IL-7 gene is driven by an MHC class II (Ealpha) promoter, using both in vitro and in vivo methods. Bone marrow cells were prepared from mice tibiae and femora by flushing out the bone marrow. For osteoclast-like cell (OCL) formation, cells were cultured with 10ng/ml of recombinant murine receptor activator of NF-κ B (rmRANKL) and 20ng/ml of recombinant murine macrophage colony stimulating factor (rmM-CSF). For osteoblast cultures, cells were seede into 6-well plate and cultured in alpha-modified minimal essential medium (alpha-MEM) containing 10% fetal bovine serum (FBS) for the first six days. On the seventh day β -glycerophpsphate (8mM), dexamethasone (10-8M) and ascorbic acid (50 μ g/ml) were added to the culture. Tibiae from transgenic and wild type mice were excised and proceeded for histology. Serial sections (5 μ m or 8 μ m thick) were stained with Goldner's trichrome stain for the measurement of static parameters.The measurements were performed in the trabecular bone area at the proximal metaphysis of the tibiae, 200 μ m from the growth plate, and equally distant from either endocortical bone. Trabecular bone volume (BV/TV) was measured using Osteomeasure software. Osteoclasts were identified on tartrate-resistant acid phosphatase (TRAP) stained serial sections. Growth plate area, trabecular area and endocortical length were measured and osteoclasts were counted using Osteomeasure software. Single cell suspensions were prepared from bone marrow nad peripheral blood. Different cell populations were identified using fluorescent labeling, followed by flow-cytometric analysis. Staining for flow-cytometry was performed usin following antibodies: anti CD45R and CD19 (B-cell development markers), CD11b (monocyte-macrophage population marker) and CD3 (T-cell surface marker). In IL-7 transgenic mice, the number of OCL cells was significantly higher than in B6 control mice. Hystomorphometric analysis of mouse tibias revealed smaller trabecular bone volume in transgenic mice compared to aged-matched controls. Smaller trabecular volume was also seen in mouse vertebra. There was a significant difference in total osteoclast number as well as in the number of epyphiseal plate osteoclasts between transgenic and wild type mice. The proportion of CD45R positive cells was increased three or four fold in bone marrow of IL-7 transgenic mice compared to wild type mice. CD19 positive population as well as CD19/CD45R positive population was increased four fold in the bone marrow of IL-7 transgenic mice. Cd11b positive cells were decreased compared to wild type bone marrow. Our results suggest a specific bone phenotype in IL-7 transgenic mice, characterized by a decreae in the trabecular bone volume, which is the result of an increased osteoclast number and consequently increased bone resorption. Further investigation is needed to explore the osteoclast functionality and to identify the possible role of CD45R positive population a osteoclast precursors.

bone phenotype; IL-7; transgenic mice

Rad je kao poster prezentiran i na skupu EMBO/HHMI Central European Scientists Meeting, održanom od 15.-17.06.2006., Dubrovnik/Cavtat, Hrvatska ; objavljen u knjizi sažetaka ; Zagreb : EMBO, HHMI, 2006., str. 81-81