Full text of DOI:10.1359/jbmr.2002.17.6.991

JOURNAL OF BONE AND MINERAL RESEARCH
Volume 17, Number 6, 2002
© 2002 American Society for Bone and Mineral Research
Tumor Necrosis Factor Modulates Apoptosis of Monocytes
in Areas of Developmentally Regulated Bone Remodeling
S. VOLEJNIKOVA,1 S.C. MARKS JR.,2 and D.T. GRAVES1
ABSTRACT
Tooth eruption is characterized by spatially segregated bone resorption along the path of eruption and bone
formation in the opposite direction. Monocyte recruitment occurs in two distinct peaks in both areas of
resorption and formation. Without such recruitment tooth eruption does not occur. The signals that regulate
this recruitment are thought to involve the expression of cytokines and chemokines. One such cytokine is
tumor necrosis factor (TNF), which can affect monocyte recruitment through the induction of chemokines and
adhesion molecules and increase their lifespan by acting as antiapoptotic cell survival signals. We examined
the latter by studying mice with targeted deletions of TNF receptors p55 and p75 (TNFRp55/p75). The results
indicate that mice that lack functional TNF receptors have a significantly reduced number of monocytes in the
apical area associated with bone formation. The reduced number of monocytes in this area can be accounted
for by an increase in apoptosis in TNFRp55؊/؊/p75؊/؊. In contrast, the number of monocytes, the rate of
monocyte apoptosis, and the formation of osteoclasts in the occlusal area associated with bone resorption
occurred independently of TNF activity. These results suggest that TNF receptor signaling can affect tooth
eruption by acting as a monocyte survival signal in some but not all areas of bone undergoing developmentally
regulated remodeling. (J Bone Miner Res 2002;17:991–997)
Key words: bone formation, bone resorption, leukocyte, macrophage, tooth eruption
Monocytes/macrophages also are thought to play a role in
some aspects of organogenesis. Recent evidence indicates
that mononuclear phagocytes are important in removing
apoptotic cells in the developing embryo.(2) They are found
in the remodeling interdigital regions of the mouse foot-
plate, suggesting a role in clearing areas of interdigital
apoptosis. Similarly, monocytes/macrophages have been
proposed to influence heart development by inducing apo-
ptosis and by clearing the cellular debris that is generated.(3)
Direct evidence establishing the role of monocytes/
macrophages in development comes from studies with
transgenic mice. In one example, transgenic mice were
generated in which mononuclear phagocytes were deleted
with a targeted diphtheria toxin, which resulted in the per-
INTRODUCTION
HE MONONUCLEAR-PHAGOCYTE system includes mono-
cytes and their precursors in the bone marrow, circulat-
T
ing monocytes, and tissue macrophages.(1) Monocytes and
macrophages play an essential role in the innate host re-
sponse, in activating acquired immune responses, and or-
chestrating the early events in response to wounding. These
cells directly participate in cellular debridement and pro-
duce growth factors and cytokines that affect the recruit-
ment of other cells, stimulate cellular proliferation, and
influence tissue remodeling.
The authors have no conflict of interest.
1Department of Periodontology and Oral Biology, Boston University School of Dental Medicine, Boston, Massachusetts, USA.
2Department of Cell Biology, University of Massachusetts Medical School, Worcester, Massachusetts, USA.
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992
VOLEJNIKOVA ET AL.
sistence of two different ocular tissues that disappeared mersed in periodate-lysine-paraformaldehyde fixation
rapidly in normal control mice.(4)
solution at 4°C for 4 h as described by McLean and Na-
Tooth eruption provides a model to study developmen- kane.(17) After fixation, specimens were washed for 15
tally regulated bone remodeling. Bone resorption (occlusal minutes each in 5, 10, and 15% glycerol in phosphate-
area) and bone formation (apical area) are localized pre- buffered saline (PBS) at 4°C. They were then decalcified at
cisely during tooth eruption and temporally synchronized 4°C for 2–4 days in 15% glycerol/EDTA solution as pre-
within alveolar bone.(5) The dental follicle, located between viously described.(18) To decrease freezing artifacts, speci-
the enamel organ and bone, initiates and regulates the pro- mens were then immersed into 30% sucrose overnight at
cess of tooth eruption.(6) It is a source of cells (such as 4°C. Then, they were snap-frozen in prechilled (Ϫ80°C)
monocytes and osteoclasts) and growth factors that mediate 2-methylbutane and stored at Ϫ80°C. Half-mandibles were
remodeling of the surrounding tissues. The presence of embedded in HISTO PREP compound (Fisher, Fair Lawn,
some of these molecules such as colony-stimulating factor NJ, USA) and cryosectioned at 5- to 6-␮m thickness. His-
(CSF) 1 appears to be required for tooth eruption.(7)
tological slides were prepared by sectioning the mandibles
Tumor necrosis factor (TNF) is a proinflammatory cyto- in a vertical plane perpendicular to the occlusal surface of
kine produced primarily by monocytes and macrophages, the first molar. This plane captured a sagittal section of the
but it also can be produced by other cell types. Recent first molar with surrounding bone and adjacent soft tissue
evidence suggests that TNF-␣ has several developmental occlusally and on the opposite side, the basal area between
roles, particularly in regulating apoptosis.(8) Although TNF developing apex and fornix of the tooth. Serial sections
induces apoptosis in many different normal and transformed were collected and numbered on precleaned, precharged
cells, it often has an antiapoptotic effect on inflammatory slides (Superfrost/Plus, ProbeOn Plus; Fisher).
cells.(9–11) TNF pro- and antiapoptotic properties are depen-
dent on the type of TNF receptor that is activated and on the
Immunohistochemistry
intracellular signaling molecules generated.(12–15)
To study recruitment of monocytes to defined areas of
interest, frozen serial sections were incubated with rat
monoclonal antibody F4/80 (American Type Culture Col-
lection [ATCC], Rockville, MD, USA), which identifies a
plasma membrane marker (CD68) on murine monocytes
and macrophages.(19,20) For a negative control, slides were
incubated with normal rat immunoglobulin G (IgG). Sam-
ples were incubated subsequently with biotinylated second-
ary antibody (rabbit anti-rat IgG, H ϩ L, mouse adsorbed)
and then with 0.3% hydrogen peroxide in methanol to
suppress endogenous peroxidase activity. An indirect im-
munoperoxidase technique (avidin-biotin-horseradish per-
oxidase) using diaminobenzidine (DAB) as chromogen
(Vector Laboratories, Burlington, CA, USA) was used to
detect primary-secondary antibody complexes. Slides were
counterstained with hematoxylin.
The study presented here was undertaken to investigate
the involvement of TNF receptor signaling in regulating
apoptosis during developmentally regulated bone remodel-
ing that occurs in tooth eruption. For this purpose, we
examined mice with genetic deletions of the TNF receptor
p55 and p75 (TNFRp55/p75) and compared the results to
control animals. The results indicate that there were signif-
icantly greater numbers of monocytes in the control than in
TNF receptor–deficient mice in areas of bone formation,
and there was no difference in areas of bone resorption.
Moreover, the decrease in monocyte number could be ac-
counted for by an increase in apoptosis. In contrast, there
were no differences in apoptosis in areas of bone resorption
in TNF receptor–deficient mice and their normal counter-
parts. Thus, during tooth eruption, TNF activity has an
antiapoptotic effect in areas of bone formation, but surpris-
ingly, no impact on apoptosis in areas of bone resorption.
This study suggests that subpopulations of monocyte are
differentially regulated by antiapoptotic factors in develop-
mentally regulated remodeling bone.
Tartrate-resistant acid phosphatase
Tartrate-resistant acid phosphatase positive (TRAPϩ)
cells were identified as described by Marks et al.(5) Oste-
oclasts were counted as TRAPϩ multinucleated cells lining
bone. The number of osteoclasts was expressed per length
of bone.
MATERIALS AND METHODS
Animals and tissue preparation
Twenty-six different litters of transgenic and wild-type
control mice were examined to establish the rate of tooth
eruption. Experimental mice with targeted gene deletions of
TNFRp55Ϫ/Ϫ/p75Ϫ/Ϫ were generously donated by the Im-
Terminal deoxynucleotidyl transferase labeling for
apoptosis detection
Apoptotic cells were detected by an in situ terminal
munex Corp. (Seattle, WA, USA) and have been described deoxynucleotidyl transferase –mediated deoxyuridine
previously.(16) Control wild-type mice with a matching ge- triphosphate (TdT)–biotin nick end labeling (TUNEL) as-
netic background (C57BL/6 ϫ 129J hybrids) were pur- say using a TACS 2 TdT kit purchased from Trevigen
chased from The Jackson Laboratory (Bar Harbor, ME, following the manufacturer’s instructions (Trevigen, Gaith-
USA). Mice were killed in a carbon dioxide chamber. Six ersburg, MD, USA). This kit detects double-strand breaks in
animals per group were analyzed for quantitative immuno- genomic DNA and identifies cells at most stages of apopto-
histochemistry. From each animal, mandibles were re- sis. As a negative control, slides were incubated with 1ϫ
sected, separated into two halves, and immediately im- PBS instead of the TdT reaction mix. Streptavidin-

TNF AND MONOCYTE APOPTOSIS
993
horseradish peroxidase was then applied for 10 minutes and
detected by TACS Blue Label. Slides were rinsed in xylene,
counterstained with Red C, and stored in minimal light
exposure to prevent fading.
Analysis
Immunostained histological slides were examined using
light microscopy and image analysis software. Two to three
sections were analyzed per specimen. Similar areas of in-
terest were subjected to analysis; quantitative changes in
monocyte recruitment as well as numbers of apoptotic cells
were evaluated in the basal (apical) area at the site of root
bifurcation, extending from bone to the enamel organ of the
tooth, which consisted largely of the dental follicle. A
similar evaluation was carried out in the occlusal area.
Histomorphometric measurements were recorded as fol-
lows: mononuclear phagocytes were counted as F4/80ϩ
cells per follicular area (cells/mm2). Apoptotic cells were
counted at 400ϫ magnification and expressed as the number
of cells per tissue area. All cell counts and measurements
were confirmed by random reanalysis of approximately
one-third of the specimens by the same examiner and by one
other independent examiner to insure consistency. The
intra- and interexaminer variation was Ͻ15%. An unpaired
t-test was used to determine significant differences (p Յ
0.05) between experimental and wild-type animals.
FIG. 1. TNF receptor signaling modulates the number of monocytes
present in areas of bone formation during tooth eruption. The lower
mandible was surgically dissected from 9-day-old newborn wild-type
mice or TNFR1Ϫ/Ϫ/R2Ϫ/Ϫ mice, fixed in paraformaldehyde, and fro-
zen. Sections were immunostained with F4/80 antibody as described in
the Materials and Methods section. Arrows point to immunopositive
monocytes.
RESULTS
Given new findings that are emerging about the role of
inflammatory mediators in development, we assessed the
importance of TNF in regulating apoptosis during tooth
eruption. To establish if there was cytokine regulation of
tissue remodeling in distinct microcompartments, we com-
pared areas of bone formation to those of bone resorption.
Based on our previous report that the highest number of
Because TNF has been shown to have an antiapoptotic
monocytes was found at day 5 and day 9, we selected these effect on monocytes, experiments were carried out to deter-
time points for analysis.(21)
mine whether this could account for the reduced number of
Histological sections of the apical area of erupting molars monocytes observed in the TNF receptor–deficient animals
from TNFRp55Ϫ/Ϫ/p75Ϫ/Ϫ and control mice at day 9 were (Fig. 3). Day 9 was selected based on data shown in Fig. 2.
tested for the presence of monocytes by immunohistochem- In the apical area containing monocytes (the dental follicle),
istry using the F4/80 antibody (Fig. 1). The predominant very few apoptotic cells were identified by the TUNEL
cell types in the dental follicle were fibroblastic and leuko- assay in wild-type animals, and there appeared to be more
cytic cells, the latter consisting almost entirely of mono- apoptotic cells in the TNFRp55Ϫ/Ϫ/p75Ϫ/Ϫgroup. Apoptotic
cytes. On day 9, there were fewer F4/80 immunostained cells had a mononuclear cell appearance as shown in the
cells in TNFRp55Ϫ/Ϫ/p75Ϫ/Ϫ mice compared with wild- middle panel of Fig. 3. It should be noted that apoptosis of
type mice. However, at the earlier time point (day 5), mononuclear leukocytes could be readily distinguished at
F4/80ϩ monocyte numbers in the control and experimental high magnification from fibroblastic cells by the appearance
groups appeared to be similar (data not shown).
of the nuclei. When quantitative measurements were made,
The number of monocytes in the apical area was quanti- the number of apoptotic cells in the apical area of the TNF
fied in the experimental and wild-type groups (Fig. 2). On receptor–ablated group was almost twice that in normal
day 5, the number of monocytes in the dental follicle was matched controls. (p Ͻ 0.05; Fig. 4).
similar in the TNFRp55Ϫ/Ϫ/p75Ϫ/Ϫ and wild-type groups.
Different observations were made in the occlusal area
However, on day 9 the lack of TNFRp55Ϫ/Ϫ/p75Ϫ/Ϫ sig- where the predominant activity is bone resorption. Here,
naling resulted in a 40% decrease in the number of mono- lack of TNF signaling had no significant effect on the
cytes compared with the control group, which was statisti- number of mononuclear phagocytes on day 5 and day 9
cally significant (p Ͻ 0.05).
(Fig. 5A). Although there were more cells undergoing ap-

994
VOLEJNIKOVA ET AL.
FIG. 2. Quantitation of monocytes in areas of bone formation during
tooth eruption. Frozen sections were immunostained with F4/80 anti-
body to identify monocytes as described in Fig. 1 and counted at 500ϫ
magnification. Each value represents the mean of six specimens Ϯ
SEM. *Statistical significance compared with the control (p Ͻ 0.05) as
determined by Student’s t-test.
optosis in the occlusal area than that in the apical, the
number of apoptotic cells in the dental follicle and adjacent
bone was comparable between the TNFRp55Ϫ/Ϫ/p75Ϫ/Ϫand
the wild-type groups (Fig. 5B). Further evidence that there
was little difference in the area of resorption between the
experimental and wild-type groups comes from measure-
ments of osteoclasts (Fig. 6). No difference was found
between the two groups.
Monocyte recruitment to the occlusal area of the dental
follicle is associated closely with tooth eruption. To estab-
lish the functional significance of the observation that leu-
kocyte apoptosis and the number of monocytes present was
similar in the experimental and control groups in the area of
bone resorption, we examined the rate of tooth eruption
(Fig. 7). The first clinical sign that a tooth has erupted is the
presence of a cusp penetrating the gingiva. The rate of tooth
eruption was determined by counting the percentage of
cusps of the mandibular first molars that were visible each
day between days 14 and 18. No difference was observed
between the TNFRp55Ϫ/Ϫ/p75Ϫ/Ϫ and the wild-type
groups. In both groups, 50% of the first molar cusps had
erupted into the oral cavity by day 15. In fact, comparable
rates of molar eruption in both groups were noted at all time
points. By day 18, the mandibular first molars had erupted
in all of the mice in both experimental and control groups.
FIG. 3. Evidence of apoptotic cells in TNFRp55Ϫ/Ϫ/p75Ϫ/Ϫ and
wild-type mice in areas of bone formation during tooth eruption.
Apoptotic cells were identified by the TUNEL assay in histological
sections taken from specimens from 9-day-old mice. Arrows point to
apoptotic cells, which are stained blue. (A) 200ϫ magnification; (B)
400ϫ magnification; (C) 200ϫ magnification.
DISCUSSION
that TNF also is an important signal for cell survival.(11,25)
TNF was first identified as a mediator that induces tumor These two distinct functions suggest different effects from
cells to undergo apoptosis.(22–24) This finding was extended TNF stimulation depending on the specific intracellular
so that it is now recognized that TNF can induce apoptosis signaling pathways induced.(15,26) In the absence of
in normal cells as well. However, recent findings indicate TNFRp55/p75 signaling we noted fewer monocytes in the

TNF AND MONOCYTE APOPTOSIS
995
FIG. 4. The number of apoptotic cells is higher in TNFRp55Ϫ/Ϫ
/
p75Ϫ/Ϫ compared with wild-type mice in areas of bone formation.
Sections described in Fig. 3 were examined for quantitative assessment
of apoptotic cells in the area of bone formation. The difference between
experimental and control groups was statistically significant (p Ͻ 0.05).
area associated with bone formation compared with normal
controls. This finding suggests that either recruitment of
monocytes into the area is TNF dependent or that in the
absence of TNF receptor signaling, cells lose a survival
signal and undergo apoptosis. The latter possibility is sup-
ported by the fact that apoptosis of leukocytes was twofold
greater in the TNFRp55Ϫ/Ϫ/p75Ϫ/Ϫ mice. Thus, mice lack-
ing functional TNF receptors exhibited higher rates of ap-
optosis in the bone-forming area, indicating loss of cell
survival signaling.
FIG. 5. The number of monocytes and apoptotic cells in TNFR1Ϫ/
Ϫ
/R2Ϫ/Ϫ is similar to wild-type mice in areas of bone resorption during
tooth eruption. (A) Monocytes were identified by immunohistochem-
istry and counted as described in Fig. 2. (B) Apoptotic cells were
measured in specimens collected on day 9 as described in Fig. 3.
Significance was established at the p Ͻ 0.05 level by Student’s t-test.
Each value represents the mean of six specimens Ϯ SEM.
Surprisingly, there was no difference in monocyte num-
bers between the TNFRp55Ϫ/Ϫ/p75Ϫ/Ϫ and wild-type mice
in the occlusal area that is associated with bone resorption. different stages of maturation have different responses to
Consistent with this was the finding that the rate of apopto- proapoptotic stimuli. Similarly, the response of differenti-
sis was unaffected by the absence of TNF signaling. In the ated monocytes to apoptotic stimuli depends on the devel-
apical area, monocytes may be regulated by TNF and there- opmental history or differentiation factors to which the cells
fore are sensitive to lack of TNF signaling. In the occlusal have been exposed previously.(28,30) Thus, the stimuli to
area TNF does not appear to play a significant role in the which monocytes/macrophages are exposed in the area of
regulation of local tissue remodeling, as suggested by the bone formation may differ from those in the area of resorp-
equivalent number of osteoclasts present, and, therefore, tion, leading to differences in the dependence on TNF as a
would not affect the presence/function of monocytes at this survival signal.
developmental site. Monocytes present in the microcom-
The rate of tooth eruption is determined by the breakdown of
partment of alveolar bone may be exposed to different bone and connective tissue along the path of eruption, which
factors than those in the microcompartment undergoing involves the recruitment of monocytes. Monocytes can con-
bone resorption. Alternatively, these compartments may tribute to this process in a number of ways. For example,
recruit monocytes of distinctly different developmental monocytes are thought to produce factors such as IL-1 that
backgrounds.
regulate tooth eruption, presumably by stimulating osteoclas-
Programmed cell death can result from exposure of cells togenesis or induction of lytic enzymes that remove dense
to proapoptotic stimuli or from the absence of antiapoptotic connective tissue. Monocytes/macrophages also could partici-
signals.(11,27–29) In particular, monocytes require specific pate through the removal of cellular debris that is likely to
signals to maintain their viability. In vitro studies have occur. Osteoclasts that form are derived from committed
shown that cultured human monocytes are rescued from precursors, which represent a distinct subpopulation of
apoptosis by numerous proinflammatory cytokines, includ- monocytic cells. Both monocytes/macrophages and com-
ing interleukin (IL)-1␣, IL-1␤, TNF-␣, granulocyte- mitted osteoclast precursors are present along the path of
macrophage (GM)-CSF, interferon-␥ (INF-␥), IL-3, and bone resorption.(6,7) That the targeted deletion of TNF re-
CSF-1.(11,28) In vitro experiments show that monocytes at ceptors had no effect on osteoclastogenesis or the rate of

996
VOLEJNIKOVA ET AL.
MCP-1 is induced principally in osteoblasts. The recruit-
ment of monocytes in close proximity to MCP-1–
expressing cells coincides temporally with peaks in MCP-1
expression.(21,34) Findings that monocytes are recruited to
distinct microcompartments associated with different pro-
cesses, bone formation in the apical area, and resorption in
the occlusal suggests that they may have a different function
in each microenvironment. The present data suggest that
TNF receptor signaling is important for maintaining mono-
cyte numbers in developmentally regulated processes but
that its influence depends on the specific microenvironment.
Thus, factors that regulate apoptosis of mononuclear phago-
cytes in these distinct microenvironments and the cells
recruited to these sites during tooth eruption require further
systematic investigation.
FIG. 6. The number of osteoclasts is similar in TNFR1Ϫ/Ϫ/R2Ϫ/Ϫ
and wild-type mice in areas of bone resorption. Osteoclasts were
identified as TRAPϩ multinucleated bone lining cells. Each value
represents the mean of six specimens Ϯ SEM. No significant difference
was found between experimental and control groups at either time point
(p Ͼ 0.05).
ACKNOWLEDGMENTS
This work was supported by grants from the National
Institute of Dental and Craniofacial Research DE07559 and
DE07444.
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Address reprint requests to:
D.T. Graves, D.D.S., D.MSc.
Boston University Medical Center
Division of Oral Biology, Room W-202
700 Albany Street
Boston, MA 02118, USA
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Cooperation of both TNF receptors in inducing apoptosis: 25, 2001; accepted September 14, 2001.