REVIEWTNFa 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,2Regis 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 andaseptic 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 cytokines may induce resorption indirectly by affecting the production of the essential osteoclast differentiation factor, receptor activator of NF-kB ligand, and/or its soluble decoy receptor, osteoprotegerin, byosteoblast/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 byboth experimental and clinical evidence. However, TNFa is not absolutely required for osteoclastogenesis, erosive arthritis, or osteolysis, as all these events could occur in the absence of TNFa andwhether TNFa promotes osteoclast formation independently of RANK signaling is still a topic ofdebate. Here we review our current understanding of the mechanisms whereby TNFa increases osteoclastogenesis 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
Osteoclasts are multinucleated cells formed by fusion of mononuclear progenitors in the monocyte/
macrophage lineage derived from the colony-forming
units granulocyte-macrophage (CFU-GM). Considerable progress has been made in our understanding ofosteoclastogenesis through cell culture techniques,1 andthe generation of transgenic and knockout mice.2 Thesestudies have identiﬁed two distinct signaling pathways
whose activation is required for osteoclastogenesis.
The ﬁrst is activated by macrophage-colony stimulating
factor (M-CSF), which signals through its receptor cFms, and the second by the ligand of receptor activatorof NF-kB (RANKL) through its receptor, RANK.3,4
Mice genetically deﬁcient in M-CSF5,6 or RANKL7,8
signaling do not form osteoclasts and thus develop
osteopetrosis. Osteoclastogenesis is also dependent
on intracellular signaling molecules downstream fromc-Fms and RANK.9–11 These include the adapter
protein, TRAF6,12,13 and the transcription factors,
AP-1,14,15 NF-kB16,17 and NFAT.18,19
The progression of osteoclast precursors through thevarious stages of differentiation from the pluripotent
hematopoietic stem cell has been characterized by using
a combination of cell surface markers, cell sorting andosteoclastogenesis assays.20 To date, the earliest identiﬁed osteoclast progenitor in bone marrow has been
phenotyped as a c-Kitþ /c-Fms /CD11b /RANK cell,which then differentiates into a c-Kitþ /c-Fmsþ /CD11bþ /RANK early stage progenitor. Stimulation of thiscell by M-CSF expressed by osteoblast/stromal cells in
the marrow advances differentiation to the c-Kit /cFmsþ / CD11bþ /RANKþ late stage progenitor, which
responds to RANKL to complete osteoclast development. RANKL not only delivers a ﬁnal differentiation
signal, but also activates osteoclasts and promotes theirsurvival.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: Brendan_Boyce@URMC.Rochester.Edu
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TNF in Inﬂammatory Arthritis
TNFa, like many other osteoclast-stimulating molecules, promotes RANKL production by stromal cells,24
and also induces its secretion by T lymphocytes,25 B
lymphocytes,26 and endothelial cells27 to induce osteoclast formation indirectly. TNFa also stimulates MCSF production by murine or human stromal cells.28
Whether TNFa promotes osteoclast formation independently of RANK signaling has been a topic ofconsiderable debate. Two independent groups demonstrated that TNFa promotes osteoclast formation invitro despite RANK signaling blockade,29,30 whileothers reported that ‘‘permissive’’ levels of RANKL arerequired for TNFa-induced osteoclastogenesis.31 These
discrepant ﬁndings may reﬂect different cell populations
and culture conditions, but the observation that TNFa
can stimulate osteoclast formation from bone marrowcells from RANK and RANKL knockout mice clearly
demonstrates RANKL/RANK independent osteoclastogenesis in vitro32 (and our unpublished observations).
Although small numbers of osteoclasts were reported
to form in vivo in response to high local concentrationsof 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
TNFa in vitro to normal or diseased states remains
unclear.
To study the effects of chronic TNFa exposure on
osteoclast precursor differentiation and the requirement of RANKL/RANK signaling in this process, weused TNFa transgenic (TNF-Tg) mice, which develop
erosive arthritis, and RANK knockout mice.33,34 In
TNF-Tg mice there is a chronic low-level expression of
the human TNFa transgene and as a result the micedevelop an erosive arthritis with features similar to
those seen in human rheumatoid arthritis (RA). Thesefeatures include inﬂammatory bone loss,35 most notably focal erosions affecting the immediate subchondralbone and bone at the joint margins where osteoclast
numbers are increased (Fig. 1). Thus, the mechanisms
whereby TNFa induces osteoclast formation can bestudied using this model.
Osteoclast and CFU-GM colony formation is increased in cultures of spleen cells from TNF-Tg compared to wild-type mice. However, a more striking
ﬁnding is that mature osteoclasts are derived from
TNF-Tg splenocytes and peripheral blood mononuclear
cells (PBMC) one day earlier than from wild-type cells.
This accelerated osteoclastogenesis could not be inhibited by TNF blockade in vitro, but was observed in cultures of splenocytes from wild-type mice injected previously with TNFa.34 From these data, we conclude
that TNFa has a priming effect on osteoclast progenitor

Fig. 1 Increased osteoclastogenesis and bone erosion in joint of
TNFa transgenic/arthritic mouse. Adjacent knee joint sections from a
6-month-old TNF transgenic mouse and a wild-type littermate were
stained with H&E (upper) and for TRAP activity (lower panels),
showing inﬂammation and thickening of the synovium (red arrows) of
the transgenic mouse, massive osteoclastogenesis (red TRAP-stained
cells, short black arrows) and erosion of bone, joint cartilage andmeniscus (long black arrows).

cells in vivo thereby increasing the number of preosteoclasts in peripheral tissues.
If our interpretation is correct, and systemic TNFa
increases the number of pre-osteoclasts in peripheral
tissues like the spleen and blood, then these cells should
be identiﬁable by phenotypic surface markers. FACS
analysis showed that spleens from TNF-Tg mice had
4 to 7-fold more CD11bþ cells than wild-type mice,
but TNFa had no effect on CD11b expression in preosteoclasts. Spleen cells isolated from TNFa-treatedmice proliferated faster in response to M-CSF compared to PBS-treated mice in vitro, but direct administration of TNFa to spleen cells did not affect their proliferation. Since osteoclast precursors differentiate frommyeloid progenitors in the bone marrow, we examined
their frequency in the bone marrow of TNF-Tg mice todetermine if chronic exposure to TNF alters the generation of pre-osteoclasts. Compared to their wild-type
littermate controls, TNF-Tg mice have signiﬁcantly
increased CD11bþ pre-osteoclasts. Furthermore, TNF
treatment of bone marrow cells from wild-type miceleads to an increase in the frequency of c-Fmsþ /CD11bþ cells and increases c-Fms mRNA expression,suggesting that TNF promotes the differentiation of
marrow osteoclast precursors thereby increasing thepool size of these cells36 (Fig. 2). TNF treatment ofCD11bþ c/Gr-1=low osteoclast precursors puriﬁed frombone marrow by FACS analysis increased their c-fms
mRNA expression 4-fold, suggesting that these cells

Keio J Med 2005; 54 (3): 127–131

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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 osteoclasts 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 development (our unpublished observations). Whether thesepre-osteoclasts actively leave the bone marrow to go toperipheral tissues and whether TNF affects this process
have yet to be determined.
Having demonstrated that transgenically-expressedand exogenous TNFa increase peripheral pre-osteoclast
frequency, we examined if this increase was reversible
via in vivo TNFa blockade with the TNFa inhibitor, etanercept. This treatment reduced the number of CD11bþ
splenocytes, and their osteoclastogenic and CFU-GM
colony formation potential to wild-type levels. Theseﬁndings37 are consistent with our observation thatpatients with RA and psoriatic arthritis have a marked
increase in the number of pre-osteoclasts in their PBMC
population compared to normal and osteoarthritis controls.38 Importantly, this increase also appears to bereversible with anti-TNFa therapy, and may be a dominant mechanism by which this treatment inhibits erosions in these patients.
We previously investigated RANK-independent
osteoclastogenesis in animal models of wear debrisinduced osteolysis39 and fracture healing,40 but foundno osteoclast formation in the absence of RANK signaling. To investigate if the effects of chronic exposure
to TNFa are dependent on RANK signaling, we treated
TNF-Tg mice with a RANK antagonist, RANK : Fc (asoluble fusion protein consisting of the extracellulardomain of RANK fused to the Fc domain of IgG19),
and crossed TNF-Tg mice with RANK-/- mice. We
found that CD11bþ cells increased in both models.
However, the elevated levels of TNFa did not compensate for the absence of RANK signaling because theprecursors were unable to differentiate into mature
osteoclasts. Furthermore, we found no erosions in micewith established arthritis that had been treated withRANK : Fc, which suggests that RANK blockade canarrest erosive disease (our unpublished observations).
Thus, chronic exposure to TNFa in vivo increases
osteoclastogenesis through two distinct mechanisms(Fig. 2) in which TNFa ﬁrst affects osteoclastogenesis at
the osteoclast precursor stage in the bone marrow bypriming these cells to differentiate into c-Fmsþ /CD11bþ /RANKþ= osteoclast progenitors via a RANKL/RANK
independent mechanism. These osteoclast precursors
then enter the blood and peripheral tissues where they
can respond to TNFa by both direct and indirect
mechanisms to become mature osteoclasts at sites ofbone resorption. For example, TNFa can induce avariety of cells, including synovial cells, T cells andosteoblast/stromal cells, to increase their expression of
RANKL, which binds to RANK on the surface of theseprecursors and induces their differentiation. TNFa canalso bind to its receptor on the surface of these precursors and directly induce their differentiation to mature osteoclasts, thus enhancing the RANKL-induced
indirect action. The dominance of RANK blockade in
the model in Figure 2 is consistent with results fromother animal models of arthritis,41,42 osteoporosis,22
hypercalcemia of malignancy43,44 and tumor metastasis
to bone.45 These ﬁndings suggest a clinical correlate
whereby patients with active erosive arthritis might
have increased numbers of CD11bþ cells with enhanced
osteoclastogenic potential in their blood. It is possiblethat patients with active disease or ‘‘ﬂares,’’ could be
identiﬁed by an increase in circulating pre-osteoclasts.
Furthermore, it is possible that patients that are nonresponsive or refractory to anti-TNF therapy could
be identiﬁed by changes in this population following
therapy.

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Acknowledgements: This work was supported by research grants
from the National Institutes of Health (PHS AR45791, AR43510,
AR48697, and AR44220).
14.

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