Lipopolysaccharide induces multinuclear cell from RAW264.7 line with increased phagocytosis activity

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Abstract

Lipopolysaccharide (LPS), an outer membrane component of Gram-negative bacteria, induces strong proinflammatory responses, including the release of cytokines and nitric oxide from macrophage. In this study, we found that a murine macrophage-derived line, RAW264.7, became multinuclear through cell-cell fusion after incubation with highly purified LPS or synthetic lipid A in the presence of Ca2+. The same cell line is known to differentiate into multinuclear osteoclast, which expresses a specific proton pumping ATPase together with osteoclast markers on stimulation by the extracellular domain of receptor activator of nuclear factor κB ligand (Toyomura, T., Murata, Y., Yamamoto, A., Oka, T., Sun-Wada, G.-H., Wada, Y. and Futai, M., 2003). The LPS-induced multinuclear cells did not express osteoclast-specific enzymes including tartrate-resistant acid phosphatase and cathepsin K. During multinuclear cell formation, the cells internalized more and larger polystyrene beads (diameter 6–15 μm) than mononuclear cells and osteoclasts. The internalized beads were located in lysosome-marker positive organelles, which were probably phagolysosomes. The LPS-induced multinuclear cell could be a good model system to study phagocytosis of large foreign bodies.

Highlights

LPS induces multinuclear cells from murine macrophage-derived RAW264.7 cells. ► The multinuclear cells are formed through cell-cell fusion in the presence of Ca2+. ► The multinuclear cells do not express osteoclast-specific enzymes. ► They internalized more and larger beads than mononuclear cells and osteoclasts.

Introduction

Lipopolysaccharide (LPS), a major component of the outer membrane of Gram-negative bacteria, is recognized by the immune system as a sign of infection. LPS induces expression of a variety of pro-inflammatory cytokines and nitric oxide producing enzyme iNOS (inducible nitric oxide synthase) in macrophage [1]. These defense responses are important for the host to survive infection. Signal transduction by the LPS receptor TLR4 (Toll-like receptor-4) has been demonstrated in detail [1], [2]. However, effects of LPS on morphology of macrophage are not studied intensively. To internalize large amount of un-necessaries including host cells damaged by strong inflammatory responses, macrophage may change their morphologies.

In response to multiple signals or cytokines, macrophages differentiate into different types of multinuclear cells. Osteoclasts are one of these cells, which are attached tightly to the bone surface, and secrete protons and lysosomal enzymes for bone resorption [3], [4], [5], [6], [7]. It is important for normal bone homeostasis to maintain equilibrium between resorption by osteoclasts and bonegenesis by osteoblasts. Reduced and increased resorbing activity of osteoclasts causes osteopetrosis and osteoporosis, respectively [8]. RANKL (receptor activator of nuclear factor κB ligand) and M-CSF (macrophage colony-stimulating factor) are required for osteoclastogenesis [9], [10]. A membrane protein, RANKL, located on the surface of osteoblasts activates osteoclast progenitors through its receptor, RANK (receptor activator of nuclear factor κB) [9]. Upon incubation of murine macrophage line RAW264.7 cells with the extracellular domain of RANKL, osteoclast-like cells were observed within three days and their number reached a plateau within seven days [11], [12].

Macrophages also become multinuclear giant cell (FBGC, foreign body giant cell) originally found internalizing orthopedic implant wear debris at the site of implantation [13], [14]. They are believed to resorb large foreign bodies, which cannot be internalized or digested by mononuclear cells [14], [15]. Bone marrow macrophages differentiate into similar multinuclear cells upon stimulation with IL4 (interleukin-4) and GM-CSF (granulocyte-monocyte colony-stimulation factor) in vitro [16].

In this study, we found that RAW264.7 cells differentiate into multinuclear cells on the addition of LPS. Sequential observations revealed that these cells were formed through cell–cell fusion in the presence of Ca2+. The average number of nuclei in this cell was about 90, similar to that of osteoclasts. The multinuclear cells induced by LPS could internalize polystyrene beads (diameter 6–15 μm) more efficiently than mononuclear cells or osteoclasts. Furthermore, the beads were located in phagolysosomes, suggesting that these multinuclear cells have increased phagocytosis activity.

Section snippets

Cell culture

RAW264.7 cells obtained from the European Collection of Cell Culture were grown in Dulbecco’s Modified Eagle Medium as previously described [11]. For multinuclear cell induction with LPS, cells were harvested by pipetting, and seeded at 8 × 105 cells per well (1.77 cm2) in Minimum Essential Medium Alpha Medium (MEMα) containing 10% fetal bovine serum, 100 units/ml penicillin, and 100 μg/ml streptomycin. Fetal bovine serum was from Sigma (St. Louis, MO) and the other reagents were purchased from

Formation of multinuclear cells from RAW264.7 cells on LPS addition

Macrophages become multinuclear during differentiation into osteoclasts, which resorb the bone matrix for bone homeostasis. Similar giant cells are formed when they internalize foreign materials, including orthopedic implant wear debris [13], [14]. Macrophages also respond to the bacterial infection, thus, it became of interest to determine whether bacterial components can induce multinuclear cells. To address this question, we incubated macrophage-derived RAW264.7 cells with highly purified E.

Discussion

In this study, we have demonstrated that murine macrophage line RAW264.7 forms multinuclear cells on the addition of E. coli or Salmonella LPS, or synthetic lipid A. Although osteoclasts and FBGCs (foreign body giant cells) are multinuclear cells, they are different from LPS-induced multinuclear cells: (1) The formation of osteoclasts or FBGC were observed 3–7 days after stimulation. On the other hand, multinuclear cells are formed within 16 h after LPS addition. This rapid formation should be

Acknowledgments

This study was supported partly by the JSPS (Japan Society for the Promotion of Science), the Terumo Life Science Foundation, and the Japan Foundation for Applied Enzymology. The support of the Hayashi Memorial Foundation for Female Natural Scientists (to M. N.-M.) is also acknowledged. We are also grateful for the support of Stanley Losinski and Vicki Thomson during the difficult time after the earthquake. We thank Drs. Mizuki Sekiya and Kiyoshi Kawasaki for assistance in phagocytosis assay

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