TNF-Α Induces Osteoclastogenesis by Direct Stimulation of Macrophages Exposed to Permissive Levels of RANK Ligand

TNF-α induces osteoclastogenesis by direct stimulation of macrophages exposed to permissive levels of RANK ligand Jonathan Lam, … , F. Patrick Ross, Steven L. Teitelbaum J Clin Invest. 2000;106(12):1481-1488. https://doi.org/10.1172/JCI11176. Article While TNF-α is pivotal to the pathogenesis of inflammatory osteolysis, the means by which it recruits osteoclasts and promotes bone destruction are unknown. We find that a pure population of murine osteoclast precursors fails to undergo osteoclastogenesis when treated with TNF-α alone. In contrast, the cytokine dramatically stimulates differentiation in macrophages primed by less than one percent of the amount of RANKL (ligand for the receptor activator of NF-κB) required to induce osteoclast formation. Mirroring their synergistic effects on osteoclast differentiation, TNF-α and RANKL markedly potentiate NF-κB and stress-activated protein kinase/c-Jun NH2-terminal kinase activity, two signaling pathways essential for osteoclastogenesis. In vivo administration of TNF-α prompts robust osteoclast formation in chimeric animals in which β-galactosidase positive, TNF-responsive macrophages develop within a TNF-nonresponsive stromal environment. Thus, while TNF-α alone does not induce osteoclastogenesis, it does so both in vitro and in vivo by directly targeting macrophages within a stromal environment that expresses permissive levels of RANKL. Given the minuscule amount of RANKL sufficient to synergize with TNF-α to promote osteoclastogenesis, TNF-α appears to be a more convenient target in arresting inflammatory osteolysis. Find the latest version: https://jci.me/11176/pdf TNF-α induces osteoclastogenesis by direct stimulation of macrophages exposed to permissive levels of RANK ligand Jonathan Lam,1,2 Sunao Takeshita,1 Jane E. Barker,3 Osami Kanagawa,1 F. Patrick Ross,1 and Steven L. Teitelbaum1 1Department of Pathology and Immunology, and 2Division of Biology and Biomedical Sciences, Washington University School of Medicine, St. Louis, Missouri, USA 3The Jackson Laboratory, Bar Harbor, Maine, USA Address correspondence to: Steven L. Teitelbaum, Washington University School of Medicine, Barnes-Jewish Hospital North, Mailstop 90-31-649, 216 S. Kingshighway Boulevard, St. Louis, Missouri 63110, USA. Phone: (314) 454-8463; Fax: (314) 454-5055; E-mail: [email protected]. Jonathan Lam and Sunao Takeshita contributed equally to this work. Received for publication August 26, 2000, and accepted in revised form November 13, 2000. While TNF-α is pivotal to the pathogenesis of inflammatory osteolysis, the means by which it recruits osteoclasts and promotes bone destruction are unknown. We find that a pure population of murine osteoclast precursors fails to undergo osteoclastogenesis when treated with TNF-α alone. In contrast, the cytokine dramatically stimulates differentiation in macrophages primed by less than one percent of the amount of RANKL (ligand for the receptor activator of NF-κB) required to induce osteoclast formation. Mirroring their synergistic effects on osteoclast differentiation, TNF-α and RANKL markedly potentiate NF-κB and stress-activated protein kinase/c-Jun NH2-terminal kinase activity, two signaling pathways essential for osteoclastogenesis. In vivo administration of TNF-α prompts robust osteoclast formation in chimeric animals in which β-galactosidase positive, TNF-responsive macrophages develop within a TNF-nonresponsive stromal environment. Thus, while TNF-α alone does not induce osteoclastogenesis, it does so both in vitro and in vivo by directly targeting macrophages within a stromal environment that expresses permissive levels of RANKL. Given the minuscule amount of RANKL sufficient to synergize with TNF-α to promote osteoclastogenesis, TNF-α appears to be a more convenient target in arresting inflammatory osteolysis. J. Clin. Invest. 106:1481–1488 (2000). Introduction Osteoclasts are bone-resorbing polykaryons derived The most prevalent form of clinically significant from macrophage or myeloid-lineage progenitors osteopenia involves chronic inflammation of bone or under the influence of RANKL, a TNF superfamily periosseous tissues by gram-negative bacteria, as seen cell-surface molecule expressed by marrow stromal in suppurative otitis media (1) and periodontitis (2, 3). cells and osteoblasts, and by activated T lymphocytes Severe osteolysis occurring as a sequela of chronic (8). Most osteoclastogenic factors, including TNF-α, infection reflects enhanced osteoclastic bone resorp- act via receptors on stromal or osteoblastic cells to tion, a process driven by host inflammatory responses enhance RANKL expression (9, 10). In contrast, sever- (4, 5). Prolonged or excessive release of cytokines such al recent studies posit that TNF-α directly stimulates as TNF-α, IL-1, IL-6, and IL-8 is central to the patho- macrophages to differentiate into osteoclasts by a genesis of inflammatory bone and joint destruction. A mechanism independent of RANKL (11, 12). Thus, the greater understanding of the molecular and cellular issue of the precise target cell(s) for the TNF-α effect mechanisms by which inflammatory cytokines and the nature of the role of TNF-α in basal and enhance osteoclastogenesis will provide insight into inflammatory osteoclastogenesis remain controversial. potential therapeutic and preventative strategies for We have examined the impact of TNF-α on osteoclast inflammatory osteopenic disorders. ontogeny. We find that TNF-α alone, at any concentra- TNF-α is among the most potent of the osteoclasto- tion, fails to induce the differentiation of murine osteo- genic cytokines produced in inflammation. TNF- clast precursors. Rather, TNF-α dramatically enhances α–induced catabolic processes are implicated in the in vitro osteoclastogenesis primed by a level of RANKL pathogenesis of conditions including rheumatoid that is insufficient to induce osteoclast formation. arthritis, orthopedic implant loosening, and other Most importantly, TNF-α stimulates osteoclastogene- forms of chronic inflammatory osteolysis (1–5). In fact, sis in circumstances both in vitro and in vivo in which TNF-α mediates lipopolysaccharide-stimulated osteo- osteoclast precursors, but not RANKL-producing stro- clastogenesis via its p55 receptor, through a mecha- mal cells, are responsive to the cytokine. Thus, TNF-α nism involving activation of NF-κB (6, 7). targets both marrow stromal cells and osteoclast pre- The Journal of Clinical Investigation | December 2000 | Volume 106 | Number 12 1481 cursors, but directly impacts the latter only in the pres- ascites, as delineated elsewhere (15). Donor marrow ence of permissive levels of RANKL. repopulation of irradiated hosts and marrow cell phe- notypes were assessed by flow cytometry. Methods Flow cytometry. Cells were labeled with antibodies at Reagents. Recombinant murine TNF-α, murine M-CSF, the manufacturers’ recommended dilutions and con- murine osteoprotegerin-human Fc chimera (OPG-Fc), ditions. Nonspecific signal was calculated and attenu- and anti-murine RANK polyclonal antibody were pro- ated by incubation with fluorochrome-conjugated sec- cured from R&D Systems Inc. (Minneapolis, Minneso- ondary antibody in the absence of primary antibody. ta, USA). Recombinant murine RANKL was prepared Labeled cells were analyzed with a FACSCalibur flow as a fusion protein with glutathione S-transferase, as cytometry system (Becton Dickinson Immunocytome- we have previously described (13), and lack of endotox- try Systems, San Jose, California, USA). in contamination was confirmed by limulus amoebo- Histochemistry. Tartrate-resistant acid phosphatase cyte lysate assay (BioWhittaker Inc., Walkersville, Mary- (TRAP) activity was identified with a commercially avail- land, USA). Microbeads for immunopurification were able kit (Sigma Chemical Co.). A quantitative TRAP obtained from Miltenyi Biotec (Auburn, California, assay was performed by addition of a colorimetric sub- USA). Anti-murine CD3, CD11b, and CD106 mAb’s strate, 5.5 mM p-nitrophenyl phosphate, in the presence were purchased from PharMingen (San Diego, Califor- of 10 mM sodium tartrate at pH 4.5. The reaction prod- nia, USA). Anti-murine c-Fms mAb (AFS98) was pre- uct was quantified by measuring optical absorbance at pared by affinity chromatography from a hybridoma 405 nm. β-galactosidase immunohistochemistry was culture provided by Shin-Ichi Nishikawa (Kyoto Uni- performed with a monoclonal primary antibody (Sigma versity, Kyoto, Japan) (14). Except where stated, all mis- Chemical Co.) and a VectaStain ABC detection system cellaneous reagents were obtained from Sigma Chem- (Vector Laboratories, Burlingame, California, USA). ical Co. (St. Louis, Missouri, USA). RT-PCR. RNA was isolated with the RNeasy Total Mice. Four- to six-week-old male C3H/HeN inbred RNA System (QIAGEN Inc., Valencia, California, USA), mice (Harlan Sprague Dawley Inc., Indianapolis, Indi- digested with deoxyribonuclease to eliminate genomic ana, USA) were employed, along with the following DNA, and characterized with the Platinum Quantita- age-matched male transgenic animals from The Jack- tive RT-PCR Thermoscript System (Life Technologies son Laboratory: mice deficient in both p55 and p75 Inc., Rockville, Maryland, USA). Briefly, 50 pg RNA was TNF receptors (B6,129S-TNFrsfla-TNFrsflb) and their reverse transcribed to cDNA using murine gene-specif- heterozygous littermates; and B6,129S-Gtrosa26 ic oligonucleotide primers designed to span exon-intron (Rosa) mice expressing a lacZ transgene. Animals were boundaries:

Load more