REVIEW
TNFa and pathologic bone resorption
Brendan F. Boyce,1,2 Ping Li,2 Zhenqiang Yao,1 Qian Zhang,1 I. Raul Badell,1 Edward M. Schwarz,2
Regis J. O Keefe2 and Lianping Xing1,2
1Department of Pathology and Laboratory Medicine, University of Rochester Medical Center, Rochester, NY, USA
2The Center for Musculoskeletal Research, University of Rochester Medical Center, Rochester, NY, USA
(Received for publication on January 18, 2005)
(Revised for publication on April 28, 2005)
(Accepted for publication on May 19, 2005)
Abstract. Chronic inflammatory bone diseases, such as rheumatoid arthritis, periodontal disease and
aseptic periprosthetic osteolysis, are characterized by bone loss around affected joints and teeth caused
by increased osteoclastic bone resorption. This resorption is mediated largely by the increased local
production of pro-inflammatory cytokines, such as tumor necrosis factor-alpha (TNFa). These cyto-
kines may induce resorption indirectly by affecting the production of the essential osteoclast differen-
tiation factor, receptor activator of NF-kB ligand, and/or its soluble decoy receptor, osteoprotegerin, by
osteoblast/stromal cells or directly by enhancing proliferation and/or activity of cells in the osteoclast
lineage. The importance of TNFa in the pathogenesis of various forms of bone loss is supported by
both experimental and clinical evidence. However, TNFa is not absolutely required for osteoclasto-
genesis, erosive arthritis, or osteolysis, as all these events could occur in the absence of TNFa and
whether TNFa promotes osteoclast formation independently of RANK signaling is still a topic of
debate. Here we review our current understanding of the mechanisms whereby TNFa increases osteo-
clastogenesis in vitro and in vivo. (Keio J Med 54 (3): 127 131, September 2005)
Key words: TNF, Cytokines, osteoclasts, NF-kB, inflammation
Regulation of Osteoclast Formation c-Fms and RANK.9 11 These include the adapter
protein, TRAF6,12,13 and the transcription factors,
Osteoclasts are multinucleated cells formed by fu- AP-1,14,15 NF-kB16,17 and NFAT.18,19
sion of mononuclear progenitors in the monocyte/ The progression of osteoclast precursors through the
macrophage lineage derived from the colony-forming various stages of differentiation from the pluripotent
units granulocyte-macrophage (CFU-GM). Consider- hematopoietic stem cell has been characterized by using
able progress has been made in our understanding of a combination of cell surface markers, cell sorting and
osteoclastogenesis through cell culture techniques,1 and osteoclastogenesis assays.20 To date, the earliest iden-
the generation of transgenic and knockout mice.2 These tified osteoclast progenitor in bone marrow has been
studies have identified two distinct signaling pathways phenotyped as a c-Kitþ/c-Fms /CD11b /RANK cell,
whose activation is required for osteoclastogenesis. which then differentiates into a c-Kitþ/c-Fmsþ/CD11bþ/
The first is activated by macrophage-colony stimulating RANK early stage progenitor. Stimulation of this
factor (M-CSF), which signals through its receptor c- cell by M-CSF expressed by osteoblast/stromal cells in
Fms, and the second by the ligand of receptor activator the marrow advances differentiation to the c-Kit /c-
of NF-kB (RANKL) through its receptor, RANK.3,4 Fmsþ/ CD11bþ/RANKþ late stage progenitor, which
Mice genetically deficient in M-CSF5,6 or RANKL7,8 responds to RANKL to complete osteoclast develop-
signaling do not form osteoclasts and thus develop ment. RANKL not only delivers a final differentiation
osteopetrosis. Osteoclastogenesis is also dependent signal, but also activates osteoclasts and promotes their
on intracellular signaling molecules downstream from survival.20 23
Reprint requests to: Dr. Brendan F. Boyce, Department of Pathology and Laboratory Medicine, University of Rochester Medical Center, 601
Elmwood Avenue, Box 626, Rochester, NY 14642, e-mail: [e-mail]
127
128 Boyce BF, et al: TNF and bone loss
TNF in Inflammatory Arthritis
TNFa, like many other osteoclast-stimulating mole-
cules, promotes RANKL production by stromal cells,24
and also induces its secretion by T lymphocytes,25 B
lymphocytes,26 and endothelial cells27 to induce osteo-
clast formation indirectly. TNFa also stimulates M-
CSF production by murine or human stromal cells.28
Whether TNFa promotes osteoclast formation inde-
pendently of RANK signaling has been a topic of
considerable debate. Two independent groups demon-
strated that TNFa promotes osteoclast formation in
vitro despite RANK signaling blockade,29,30 while
others reported that   permissive  levels of RANKL are
required for TNFa-induced osteoclastogenesis.31 These
discrepant findings may reflect different cell populations
Fig. 1 Increased osteoclastogenesis and bone erosion in joint of
and culture conditions, but the observation that TNFa
TNFa transgenic/arthritic mouse. Adjacent knee joint sections from a
can stimulate osteoclast formation from bone marrow
6-month-old TNF transgenic mouse and a wild-type littermate were
cells from RANK and RANKL knockout mice clearly
stained with H&E (upper) and for TRAP activity (lower panels),
demonstrates RANKL/RANK independent osteoclas- showing inflammation and thickening of the synovium (red arrows) of
the transgenic mouse, massive osteoclastogenesis (red TRAP-stained
togenesis in vitro32 (and our unpublished observations).
cells, short black arrows) and erosion of bone, joint cartilage and
Although small numbers of osteoclasts were reported
meniscus (long black arrows).
to form in vivo in response to high local concentrations
of TNF delivered locally over the calvarial bones of
RANK-/- mice, RANK independent osteoclastogenesis
under physiological conditions in vivo has not been
demonstrated. Thus, the relevance of this action of cells in vivo thereby increasing the number of pre-
TNFa in vitro to normal or diseased states remains osteoclasts in peripheral tissues.
unclear. If our interpretation is correct, and systemic TNFa
To study the effects of chronic TNFa exposure on increases the number of pre-osteoclasts in peripheral
osteoclast precursor differentiation and the require- tissues like the spleen and blood, then these cells should
ment of RANKL/RANK signaling in this process, we be identifiable by phenotypic surface markers. FACS
used TNFa transgenic (TNF-Tg) mice, which develop analysis showed that spleens from TNF-Tg mice had
erosive arthritis, and RANK knockout mice.33,34 In 4 to 7-fold more CD11bþ cells than wild-type mice,
TNF-Tg mice there is a chronic low-level expression of but TNFa had no effect on CD11b expression in pre-
the human TNFa transgene and as a result the mice osteoclasts. Spleen cells isolated from TNFa-treated
develop an erosive arthritis with features similar to mice proliferated faster in response to M-CSF com-
those seen in human rheumatoid arthritis (RA). These pared to PBS-treated mice in vitro, but direct adminis-
features include inflammatory bone loss,35 most nota- tration of TNFa to spleen cells did not affect their pro-
bly focal erosions affecting the immediate subchondral liferation. Since osteoclast precursors differentiate from
bone and bone at the joint margins where osteoclast myeloid progenitors in the bone marrow, we examined
numbers are increased (Fig. 1). Thus, the mechanisms their frequency in the bone marrow of TNF-Tg mice to
whereby TNFa induces osteoclast formation can be determine if chronic exposure to TNF alters the gener-
studied using this model. ation of pre-osteoclasts. Compared to their wild-type
Osteoclast and CFU-GM colony formation is in- littermate controls, TNF-Tg mice have significantly
creased in cultures of spleen cells from TNF-Tg com- increased CD11bþ pre-osteoclasts. Furthermore, TNF
pared to wild-type mice. However, a more striking treatment of bone marrow cells from wild-type mice
finding is that mature osteoclasts are derived from leads to an increase in the frequency of c-Fmsþ/
TNF-Tg splenocytes and peripheral blood mononuclear CD11bþ cells and increases c-Fms mRNA expression,
cells (PBMC) one day earlier than from wild-type cells. suggesting that TNF promotes the differentiation of
This accelerated osteoclastogenesis could not be inhib- marrow osteoclast precursors thereby increasing the
ited by TNF blockade in vitro, but was observed in cul- pool size of these cells36 (Fig. 2). TNF treatment of
tures of splenocytes from wild-type mice injected pre- CD11bþc/Gr-1 =low osteoclast precursors purified from
viously with TNFa.34 From these data, we conclude bone marrow by FACS analysis increased their c-fms
that TNFa has a priming effect on osteoclast progenitor mRNA expression 4-fold, suggesting that these cells
Keio J Med 2005; 54 (3): 127 131 129
Fig. 2 RANK dependent and independent mechanisms of TNF-mediated osteoclastogenesis. During the early stages of osteoclastogenesis,
TNF increases the pool size of marrow osteoclast precursors by promoting their proliferation and differentiation in response to M-CSF and by
stimulating c-Fms expression, which is independent of the RANK pathway. These osteoclast precursors then differentiate into mature osteo-
clasts in the presence of RANKL, and this process is accelerated by TNF. The role of TNF at this later stage of osteoclast differentiation is
RANKL/RANK dependent.
express TNF receptors at this stage in their develop- with established arthritis that had been treated with
ment (our unpublished observations). Whether these RANK : Fc, which suggests that RANK blockade can
pre-osteoclasts actively leave the bone marrow to go to arrest erosive disease (our unpublished observations).
peripheral tissues and whether TNF affects this process Thus, chronic exposure to TNFa in vivo increases
have yet to be determined. osteoclastogenesis through two distinct mechanisms
Having demonstrated that transgenically-expressed (Fig. 2) in which TNFa first affects osteoclastogenesis at
and exogenous TNFa increase peripheral pre-osteoclast the osteoclast precursor stage in the bone marrow by
frequency, we examined if this increase was reversible priming these cells to differentiate into c-Fmsþ/CD11bþ/
via in vivo TNFa blockade with the TNFa inhibitor, eta- RANKþ= osteoclast progenitors via a RANKL/RANK
nercept. This treatment reduced the number of CD11bþ independent mechanism. These osteoclast precursors
splenocytes, and their osteoclastogenic and CFU-GM then enter the blood and peripheral tissues where they
colony formation potential to wild-type levels. These can respond to TNFa by both direct and indirect
findings37 are consistent with our observation that mechanisms to become mature osteoclasts at sites of
patients with RA and psoriatic arthritis have a marked bone resorption. For example, TNFa can induce a
increase in the number of pre-osteoclasts in their PBMC variety of cells, including synovial cells, T cells and
population compared to normal and osteoarthritis con- osteoblast/stromal cells, to increase their expression of
trols.38 Importantly, this increase also appears to be RANKL, which binds to RANK on the surface of these
reversible with anti-TNFa therapy, and may be a dom- precursors and induces their differentiation. TNFa can
inant mechanism by which this treatment inhibits ero- also bind to its receptor on the surface of these pre-
sions in these patients. cursors and directly induce their differentiation to ma-
We previously investigated RANK-independent ture osteoclasts, thus enhancing the RANKL-induced
osteoclastogenesis in animal models of wear debris- indirect action. The dominance of RANK blockade in
induced osteolysis39 and fracture healing,40 but found the model in Figure 2 is consistent with results from
no osteoclast formation in the absence of RANK sig- other animal models of arthritis,41,42 osteoporosis,22
naling. To investigate if the effects of chronic exposure hypercalcemia of malignancy43,44 and tumor metastasis
to TNFa are dependent on RANK signaling, we treated to bone.45 These findings suggest a clinical correlate
TNF-Tg mice with a RANK antagonist, RANK : Fc (a whereby patients with active erosive arthritis might
soluble fusion protein consisting of the extracellular have increased numbers of CD11bþ cells with enhanced
domain of RANK fused to the Fc domain of IgG19), osteoclastogenic potential in their blood. It is possible
and crossed TNF-Tg mice with RANK-/- mice. We that patients with active disease or   flares,  could be
found that CD11bþ cells increased in both models. identified by an increase in circulating pre-osteoclasts.
However, the elevated levels of TNFa did not compen- Furthermore, it is possible that patients that are non-
sate for the absence of RANK signaling because the responsive or refractory to anti-TNF therapy could
precursors were unable to differentiate into mature be identified by changes in this population following
osteoclasts. Furthermore, we found no erosions in mice therapy.
130 Boyce BF, et al: TNF and bone loss
Acknowledgements: This work was supported by research grants JM, Paige CJ, Lacey DL, Dunstan CR, Boyle WJ, Goeddel DV,
from the National Institutes of Health (PHS AR45791, AR43510, Mak TW: TRAF6 deficiency results in osteopetrosis and defec-
AR48697, and AR44220). tive interleukin-1, CD40, and LPS signaling. Genes Dev 1999; 13:
1015 1024
14. Johnson RS, Spiegelman BM, Papaioannou V: Pleiotropic effects
of a null mutation in the c-fos proto-oncogene. Cell 1992; 71:
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