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SAT0068 Osteoclastogenic and osteoblastogenic potential of hematopoietic/stromal cells in collagen induced arthritis
  1. M. Ikic1,
  2. E. Lazić Mosler2,
  3. N. Kovačić2,
  4. A. Marušić3,
  5. D. Grčević4
  1. 1University Hospital “Sveti Duh”
  2. 2Department of Anatomy, Zagreb University School of Medicine, Zagreb
  3. 3Department of Anatomy, Split University School of Medicine, Split
  4. 4Department of Physiology and Immunology, Zagreb University School of Medicine, Zagreb, Croatia

Abstract

Background Collagen induced arthritis (CIA) is a mouse model for human rheumatoid arthritis (RA). Based on the observation that prolonged activation of the immune system causes bone loss we hypothesize that chronic inflammatory and immune responses promote osteoresorption by affecting osteoclast differentiation and/or altering their recruitment and homing. In adittion, changes in osteoblast differentiation and activity may also contribute to insufficient bone formation associated with bone loss in RA.

Objectives Our aim was to characterize the osteoclastogenic potential of bone marrow- and peripheral-hematopoietic cells, and osteoblastogenic potential of bone marrow-derived stromal cells.

Methods C57BL/6 mice were immunized with chicken type II collagen (CII). Circulating levels of anti-collagen antibodies IgG1 and IgG2a were determined in individual sera from nonarthritic and arthritic mice by ELISA. Hematopoietic cells from different sources (bone marrow, homogenized bone shafts, spleen, peripheral blood) were cultured with RANKL and M-CSF to stimulate osteoclast (OCL) differentiation. OCLs were detected as TRAP-positive multinucleated cells with 3 or more nuclei per cell. Osteoblast differentiation of bone marrow cells was induced by the addition of ascorbic acid, dexamethasone and β-glycerophosphate and analyzed by alkaline phosphatase (AP) activity and AP histochemistry. For flow-cytometry, a series of hematopoietic markers were used to characterize osteoclast progenitor (B220, CD3, NK1.1, CD115, CD117, CD11b), myeloid and lymphoid populations. Gene expression analysis for inflammatory, osteoclast and osteoblast specific genes was performed by qPCR.

Results The mice that were immunized with CII develop arthritis with incidence of almost 100% and average clinical score 10±2.6. Anti-collagen antibodies IgG1 and IgG2a were only detected in arthritic group of mice. The number of differentiated OCLs from peripheral blood, spleen and bone-shaft derived cells was significantly higher (p<0.01, Student t-test) in CIA than in control mice. There was no significant difference in OCL numbers from bone marrow- derived cells. AP activity of osteoblast differentiated from bone marrow stromal cells of arthritic mice was significantly higher than AP activity of those derived from bone marrow stromal cells of nonarthritic mice. Flow cytometry showed more CD115+ cells within both CD11b-/low and CD11b+ populations in the periphery, indicating increased number of osteoclast progenitors in CIA compared with control mice. In addition, myeloid and B-lymphoid populations increased whereas NK cell population decreased in CIA. Gene expression analysis showed increased expression of IL-17 and cFms in blood cells and decreased expression of Runx2 in bone tissue.

Conclusions Our results indicate that the peripheral-hematopoietic cells but not the bone marrow-hematopoietic cells of mice with CIA have higher number of OCL progenitors and greater osteoclastogenic potential than control mice.

Disclosure of Interest None Declared

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