Innate Immune-Like Function of Osteogenic Cells and Their Effects on Inflammatory Osteolysis

Links

Abstract

The immune response is an essential defense mechanism that protects the human body from foreign infection. Nevertheless, excessive immune response results in high levels of inflammation leading to destruction of healthy tissues. In orthopaedic fields, this duality of the immune response has been a challenge to the success of arthroplasties. Wear particle-induced inflammatory osteolysis is considered a major culprit of implant failure. Resident osteogenic bone cells, such as osteoprogenitors and osteoblasts, are exposed to wear particles. Although osteogenic cells are responsible for the initial osseointegration of implants and ongoing bone regeneration, their response to wear particles has been underestimated. Thus, the goal of this dissertation is to explore the immune mechanisms of osteogenic cells and to identify the role of osteogenic cells in particle mediated osteolysis.

To meet this end, we evaluated the immune capacity of osteogenic cells by exploring their ability to phagocytose wear particles and subsequently express pro-inflammatory cytokines. We developed a customized JAVA program and confocal microscopy methodology to quantify the phagocytic activity of osteogenic cells. Osteoprogenitors and osteoblasts were able to phagocytose Titanium (Ti) particles with aggressive actin remodeling. The actin remodeling to engulf particles activated ERK-CEBP/b pathway leading to Cox2 and IL6 gene expression. Interestingly, equibiaxial strain also increased inflammatory gene expression such as MCSF, IL6, and Cox2 through the ERK pathway. Physiological and super-physiological levels of strain were applied to osteogenic cells and macrophage-like cells via Flexcell system. Super-physiological strain exaggerated Ti particle induced inflammatory gene expression from osteogenic cells, while macrophage-like cells were not affected by strain. Taken together, these data suggest actin and ERK-CEBP/b signaling mediates phagocytosis-induced innate immune responses of osteogenic cells.

Next, we confirmed the role of osteogenic cells in inflammatory osteolysis. Although we observed that osteogenic cells secrete inflammatory cytokines after phagocytosis of wear particles, it was difficult to discern whether osteogenic cells have a major role in inflammatory osteolysis because there are a multitude of cells exposed to wear particles at the site of bone implant. Thus, we developed an osteogenic cell line specific ERK-dysfunctional mouse using an osterix-promoter-driven CRE-loxp system (CRE/dn-MEK1). An in vivo mouse calvaria model was utilized to induce inflammatory osteolysis. Ti particles were implanted on top of pericranium layer without invasive incision. This approach allowed observation of osteogenic cell responses to Ti particles in the pericranium. With this model, we observed severe calvarial osteolysis with increased osteoclastogenesis and pro-inflammatory cytokine release of IL6, PGE2 and MCSF. Significantly decreased inflammatory cytokine release and macrophage migration were observed in the CRE/dn-MEK1 mouse in in vivo mouse calvaria model and in vitro experiments. Similar trends were detected in mouse calvaria treated with AZD6244, a potent ATP-uncompetitive inhibitor of MAPK/ERK kinase.

In summary, this study supports hypothesis that (1) osteogenic cells are able to initiate inflammatory responses through well established innate immune function and (2) the ERK pathway could be a clinically important therapeutic target for preventing inflammatory osteolysis.

Cited Works (19)

Year	Entry
1997	Ducy P, Zhang R, Geoffroy V, Ridall AL, Karsent G. Osf2/Cbfa1: a transcriptional activator of osteoblast differentiation. Cell. May 30, 1997;89(5):747-754.
1999	Burger EH, Klein-Nulend J. Mechanotransduction in bone: role of the lacunocanalicular network. FASEB J. May 1999;12(9001):S101-S112.
2003	Boyle WJ, Simonet WS, Lacey DL. Osteoclast differentiation and activation. Nature. May 15, 2003;423(6937):337-342.
1999	Suda T, Takahashi N, Udagawa N, Jimi E, Gillespie MT, Martin TJ. Modulation of osteoclast differentiation and function by the new members of the tumor necrosis factor receptor and ligand families. Endocr Rev. June 1999;20(3):345-357.
1998	Lacey DL, Timms E, Tan H-L, Kelley MJ, Dunstan CR, Burgess T, Elliott R, Colombero A, Elliott G, Scully S, Hsu H, Sullivan J, Hawkins N, Davy E, Capparelli C, Eli A, Qian Y-X, Kaufman S, Sarosi I, Shalhoub V, Senaldi G, Guo J, Delaney J, Boyle WJ. Osteoprotegerin ligand is a cytokine that regulates osteoclast differentiation and activation. Cell. April 17, 1998;93(2):165-176.
2010	Kramer I, Halleux C, Keller H, Pegurri M, Gooi JH, Weber PB, Feng JQ, Bonewald LF, Kneissel M. Osteocyte Wnt/β-catenin signaling is required for normal bone homeostasis. Mol Cell Biol. June 15, 2010;30(12):3071-3085.
1998	Yasuda H, Shima N, Akagawa NN, Yamaguchi K, Kinosaki M, Mochizuki S-I, Omoyasu AT, Yano K, Goto M, Murakami A, Tsuda E, Morinaga T, Igashio KH, Udagawa N, Tkahashi N, Suda T. Osteoclast differentiation factor is a ligand for osteoprotegerin/osteoclastogenesis-inhibitory factor and is identical to TRANCE/RANKL. Proc Natl Acad Sci USA. March 31, 1998;95(7):3597-3602.
1998	Turner CH, Pavalko FM. Mechanotransduction and functional response of the skeleton to physical stress: the mechanisms and mechanics of bone adaptation. J Orthop Sci. November 1998;3(6):346-355.
2002	Takayanagi H, Kim S, Koga T, Nishina H, Isshiki M, Yoshida H, Saiura A, Isobe M, Yokochi T, Inoue J-i, Wagner EF, Mak TW, Kodama T, Taniguchi T. Induction and activation of the transcription factor NFATc1 (NFAT2) integrate RANKL signaling in terminal differentiation of osteoclasts. Dev Cell. December 1, 2002;3(6):889-901.
1995	Duncan RL, Turner CH. Mechanotransduction and the functional response of bone to mechanical strain. Calcif Tiss Int. December 1995;57(5):344-358.
1985	Rubin CT, Lanyon LE. Regulation of bone mass by mechanical strain magnitude. Calcif Tiss Int. 1985;37(4):411-417.
2002	Kalajzic I, Kalajzic Z, Kaliterna M, Gronowicz G, Clark SH, Lichtler AC, Rowe D. Use of type I collagen green fluorescent protein transgenes to identify subpopulations of cells at different stages of the osteoblast lineage. J Bone Miner Res. January 2002;17(1):15-25.
1997	Komori T, Yagi H, Nomura S, Yamaguchi A, Sasaki K, Deguchi K, Shimizu Y, Bronson RT, Gao Y-H, Inada M, Sato M, Okamoto R, Kitamura Y, Yoshiki S, Kishimoto T. Targeted disruption of Cbfa1 results in a complete lack of bone formation owing to maturational arrest of osteoblasts. Cell. May 30, 1997;89(5):755-764.
2002	Nakashima K, Zhou X, Kunkel G, Zhang Z, Deng JM, Behringer RR, de Crombrugghe B. The novel zinc finger-containing transcription factor osterix is required for osteoblast differentiation and bone formation. Cell. January 11, 2002;108(1):17-29.
1993	Bergmann G, Graichen F, Rohlmann A. Hip joint loading during walking and running, measured in two patients. J Biomech. August 1993;26(8):969-990.
2007	Tatsumi S, Ishii K, Amizuka N, Li M, Kobayashi T, Kohno K, Ito M, Takeshita S, Ikeda K. Targeted ablation of osteocytes induces osteoporosis with defective mechanotransduction. Cell Metab. June 2007;5(6):464-475.
1998	Haapasalo H, Kannus P, Sievänen H, Pasanen M, Uusi-Rasi K, Heinonen A, Oja P, Vuori I. Effect of long-term unilateral activity on bone mineral density of female junior tennis players. J Bone Miner Res. February 1998;13(2):310-319.
1995	Burger EH, Klein-Nulend J, Van Der Plas A, Nijweide PJ. Function of osteocytes in bone: their role in mechanotransduction. J Nutr. July 1995;125(7)(suppl):S2020-S2023.
1990	Udagawa N, Takahashi N, Akatsu T, Tanaka H, Sasaki T, Nishihara T, Koga T, Martin TJ, Suda T. Origin of osteoclasts: mature monocytes and macrophages are capable of differentiating into osteoclasts under a suitable microenvironment prepared by bone marrow-derived stromal cells. Proc Natl Acad Sci USA. September 1, 1990;87(18):7260-7264.