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IMMUNOBIOLOGY
From the Division of Bone and Mineral Diseases,
Washington University School of Medicine and Barnes-Jewish Hospital, St
Louis, MO, and Division of Hematology, University of Calgary, Calgary,
Canada.
In unstimulated conditions osteoclast renewal occurs as a result of
the stromal cell production of the key osteoclastogenic factors,
receptor activator of NFkB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF). Inflammation is known to cause increased osteoclastogenesis; however, the mechanisms responsible for
this phenomenon are poorly understood. We now show that
interleukin-1 (IL-1) and tumor necrosis factor alpha (TNF Activated T cells have long been associated with
the increased osteoclast (OC) formation and the accelerated bone
resorption characteristic of inflammatory conditions.1-3
Recently, evidence has accumulated that suggests that T cells stimulate
OC formation by producing the key osteoclastogenic cytokine, receptor
activator of NFkB ligand (RANKL),4,5 also known as
ODF,6 OPGL,7 and TRANCE.8
However, the mechanism(s) leading to up-regulation of T-cell RANKL
production under inflammatory conditions remains unknown, although it
is likely induced by other inflammatory cytokines. One such factor is
interleukin 7 (IL-7),9,10 a cytokine that has previously
been demonstrated to induce bone loss through increased osteoclastogenesis when administered in mice.11 Further
attesting to the relevance of IL-7 for the regulation of bone
resorption is the finding that IL-7 receptor-knockout mice show greatly
increased femoral trabecular bone volume compared with wild-type and
heterozygous littermates.11 The mechanism by which IL-7, a
product of bone marrow stromal cells (SCs),12 leads to
increased osteoclastogenesis and bone resorption remains to be
established. IL-7 is known to be a powerful inducer of both B- and
T-lymphopoiesis.11,13 As B-lymphopoiesis is also
up-regulated by E2 deficiency, a condition known to lead to bone loss,
it has been suggested that increased B-lymphopoiesis may be responsible
for the elevated osteoclastogenesis and bone loss associated with
IL-7.11 In support of this hypothesis is the recent report
that B220+ pre-B cells may be capable of differentiating
into OC precursors.14 However, as yet, no formal
demonstration that this phenomenon accounts for IL-7-induced bone loss
has been published. The mechanism of IL-7-induced OC formation and the
cell populations involved thus remain uncertain.
In this study, we demonstrate that IL-1 and tumor necrosis factor alpha
(TNF These findings suggest that IL-7 may be an important mediator of the
osteoclastogenesis and bone loss in inflammatory conditions such as
rheumatoid arthritis and periodontitis.
All reagents were purchased from the Sigma Chemical Corporation
(St Louis, MO), unless otherwise indicated. The rhIL-1 Generation of human osteoclasts
Isolation and depletion of specific cell populations
As immunomagnetic beads are phagacytosed by monocytes and cannot be removed, monocytes (a source of early OC precursors) were semipurified, after immunomagnetic antibody depletion of B cells (an adherent cell population), by adhesion onto plastic dishes for 3 hours, and nonadherent cells removed by 3 washes with phosphate-buffered saline (PBS). These populations were found to respond in the same manner as monocytes positively selected by CD14 immunomagnetic bead and were found to be greater than or equal to 64% CD14+ and greater than or equal to 99% T cells and B cells depleted as assessed by flow cytometry. Purified monocytes were plated at a density of 1 × 105
cells per well alone or with 1 × 105 purified T cells or
B cells in a final volume of 0.5 mL using 48-well plates and cultured
in In some experiments, purified monocytes (1 × 105 cells per well) received T-cell-conditioned medium that was derived from a 4× concentration of T cells (4 × 105 cells/500 µL) and added at 25% final volume (125 µL) representing a 1× final dose. Immunohistochemistry Immunohistochemical staining for V and  3 integrin
subunits, pp60c-src, cathepsin K, and for antigen to the
anti-OC antibody 121F was conducted as previously
described.16 Briefly, cells were grown in 48-well plates
and washed 3 times with PBS before fixation. Cells were fixed in
acetone:methanol (50%:50%, v/v) for 5 minutes and washed twice with
PBS. Fixed cells were incubated with primary antibody (5 µg/mL) in
PBS containing 3% bovine serum albumin (BSA) at 4°C for 18 hours.
Wells were washed 3 times in PBS for 5 minutes with shaking. Cells were
incubated with secondary antibody (antimouse IgG-biotin conjugate), in
PBS containing 3% BSA for 2 hours at room temperature, washed 3 times,
and incubated with avidin-peroxidase, in PBS containing 3% BSA for 40 minutes at room temperature. After 3 washes, color was developed using
4-chloronapthol, (0.03% in 0.05 mol/L Tris-HCl, pH 7.6, 0.1%
H2O2). Wells were washed with water after
several minutes to stop the staining. IgG isotype control was used to
assess nonspecific staining.
Calcitonin receptor analysis Expression of calcitonin receptors was assessed by autoradiography using 125I-labeled salmon calcitonin (7.4 × 107 MBq/mmol [2000 Ci/mmol]; Amersham Pharmacia Biotech, Arlington Heights, IL), as previously described.15 Briefly, mature OCs were cultured as described above, washed twice in PBS to remove nonadherent cells and labeled with 0.2 nmol/L 125I-calcitonin for 1 hour at room temperature. Two hundred-fold excess unlabeled salmon calcitonin was used to assess nonspecific binding. After washing, the cells were fixed, stained for TRAP and coated with LM-1 photographic emulsion (Amersham International, Arlington Heights, IL), and stored for 3 weeks at 4°C before developing.Resorption pit assays The ability of OCs to resorb pits was assessed by generating mature OCs (as described above) and transplanting onto whale dentine slices, in 48-well plates. After 2 to 5 days of culture, dentine slices were washed with PBS and incubated for 18 hours in 0.25 mol/L ammonium hydroxide, followed by sonication in PBS for 30 seconds. Dentine slices were washed with PBS and stained in toluidine blue (2% wt/vol) in PBS for 5 minutes, and pits photographed under light microscopy using an inverted phase contrast microscope (Olympus Optical Co, Tokyo, Japan).Measurement of human interleukin-7 production by purified stromal cells and osteoblasts Human stromal cells and osteoblasts were purified and cultured as previously described.17 Cultures were stimulated with IL-1 (1 ng/mL), TNF (20 ng/mL), or IL-1 + TNF. Cytokine
concentrations were selected on the basis of maximal stimulation of
IL-6 production by these cells, as determined
previously.17 IL-7 secreted into the culture supernatant
was measured by commercial enzyme-linked immunosorbent assay (ELISA)
(Quantikine HS, R&D Systems).
Macrophage colony-stimulating enzyme-linked immunosorbent assay M-CSF was measured in unstimulated, IL-7 (2.5 ng/mL) stimulated and IL-1 + TNF (10 ng/mL each) stimulated culture supernatants from purified T cells after 48 hours of incubation, using a commercial ELISA (Quantikine, R&D Systems)
Semiquantitative reverse transcriptase-polymerase chain reaction for receptor activator of NFkB ligand Total RNA from unstimulated and IL-7-stimulated T cells was isolated using Trizole Reagent (Gibco BRL, Gaithersburg, MA), according to the manufacturer's instructions. Semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) was conducted using Ready-To-Go RT-PCR beads (Amersham Pharmacia Biotech, Piscataway, NJ), according to the manufacturer's instructions. Briefly, 2 µg of total RNA was reverse transcribed using a pd(T)12-18 first strand primer for 30 minutes at 42°C, before PCR. The forward RANKL primer used was 5'-ATGCGCCGCGCCAGCAGAGACTACA, and the reverse primer 5'-ATCTATATCTCGAACTTTAAAAGCC. Commercial human GAPDH amplimer primers (Clontech, Palo Alta, CA) were used as internal standards. Genomic contamination was assessed by heat inactivation of the reverse transcriptase before RT-PCR. RANKL was subjected to 40 cycles of PCR and found to be in the exponential amplification range up to 45 cycles. GAPDH was cycled for 30 rounds and was exponential up to 35 cycles. PCR products were separated on 1% agarose gels and photographed under ultraviolet excitation after ethidium bromide staining.Metabolic labeling for detection of soluble receptor activator of NFkB ligand T cells were metabolically labeled as previously described.18 Briefly, cells were labeled overnight with 1.85 MBq (50 µCi) 35S-labeled methionine and cysteine (EXPRESS label, Amersham Pharmacia Biotech) and then stimulated for 24 hours with IL-7 (2.5 ng/mL). Supernatant was immunoprecipitated using antibody against human RANKL (R&D Systems) and protein G beads. The immunoprecipitated material was recovered by boiling in sample loading buffer, and separated by SDS-PAGE. Gels were dried and exposed to autoradiography film (Kodak XAR; Eastman Kodak Company, Rochester, NY).Macrophage colony-stimulating factor neutralization For M-CSF neutralization experiments, 20 µg/mL of polyclonal M-CSF antibody (R&D Systems) was added to wells before addition of IL-7-stimulated T-cell supernatant. This concentration of antibody was found to completely neutralize OC formation induced by RANKL and 500 ng/mL of rhM-CSF, a dose 50-fold higher than the concentration of soluble M-CSF estimated to be present in T-cell supernatants by ELISA. In addition, monocytes treated with M-CSF and anti-M-CSF antibody were found to be dead after 7 days in culture compared with greater than 95% viability with M-CSF-stimulated cells alone, assessed by trypan blue dye exclusion.Osteoprotegerin neutralization For osteoprotegerin (OPG) neutralization experiments, OPG (R&D Systems) was added to wells before the addition of IL-7-stimulated T-cell supernatant at a concentration ranging from 1 to 4 µg/mL. The lowest concentration of inhibitor (1 µg/mL) was found to completely neutralize OC formation induced by a saturating dose of rhRANKL (100 ng/mL) and 50 ng/mL of rhM-CSF, verifying its biologic activity.Statistical analysis Group mean values were compared by unpaired 2-tailed Student t test.
To investigate the enhanced production of IL-7 as occurs under
inflammatory conditions, we measured the secretion of IL-7 by purified
human bone marrow stromal cells and in purified human osteoblasts
before and after IL-1 and TNF
To evaluate the mechanism of IL-7-mediated osteoclastogenesis, in the absence of the confounding influence of stromal cells, we used human PBSCs, a mixed population of mononuclear cells containing monocytes/macrophages, early OC precursors of the monocytic lineage, and T- and B-lymphocytes. PBSCs do not contain SCs or SC precursors.15 When cultured in vitro with IL-1, IL-3, and GM-CSF, PBSCs generate large numbers of authentic bone resorbing OCs within 21 days of culture.15 IL-7 has previously been reported to induce proliferation of
T-lymphocytes13,19 and to induce bone loss in
mice11 through an undefined mechanism. We thus tested the
effects of rhIL-7 on OC formation in PBSC cultures. The data show that
rhIL-7 stimulates formation of OC dose dependently and within 7 to 10 days of culture (Figure 2). These cells
were found to display numerous markers of mature OCs, including TRAP,
pit resorption, calcitonin receptor, the integrin subunits
As T cells,13,19 B cells,13 and
monocytes21 are all known to respond to IL-7, we examined
which cell population(s) were responsible for transducing the
osteoclastogenic activity of IL-7. To achieve this aim, the ability of
IL-7 to induce OCs in purified monocytes alone or with monocytes
containing purified B cells or T cells was tested. The data show that
neither monocytes alone nor monocytes with B cells were capable of
supporting significant OC formation in either the presence or absence
of IL-7 (Figure 4). However, in the
presence of IL-7, mixed monocytes and T cells resulted in a large
induction of OC formation. In addition, conditioned medium (CM) from
IL-7-stimulated T cells (25% vol/vol) was also capable of inducing
substantial OC formation. The data thus suggest that the ability of
IL-7 to generate OCs in this system is related to T-cell-secreted
factors rather than to cells derived from the B-lineage, as has
previously been suggested.11 Identical results were
obtained using T cells purified from unmobilized human PBMCs rather
than from PBSCs (data not shown).
To investigate possible mechanisms by which IL-7-stimulated T cells
could induce OC formation, we evaluated the ability of IL-7 to induce
the key osteoclastogenic factor RANKL on T cells isolated from PBSCs.
Using semiquantitative RT-PCR, IL-7 was found to up-regulate RANKL
expression (Figure 5A). No RANKL
expression was observed by unstimulated T cells. Identical results were
obtained using T cells purified from unmobilized human PBMCs rather
than from PBSCs (data not shown).
Because we have identified that substantial osteoclastogenic activity is present in IL-7 stimulated T-cell CM, we conducted a metabolic labeling experiment to identify the presence of soluble RANKL. The data show (Figure 5B) the immunoprecipitation by anti-RANKL antibody, of a 26 kd protein from IL-7-stimulated T-cell CM. This protein corresponds exactly to the reported size of soluble RANKL.7,18 RANKL was not detected in unstimulated T-cell CM. Because RANKL stimulates OC formation only when OC precursors are also
stimulated with M-CSF,7,22 T cells could facilitate OC
formation by secreting M-CSF. To investigate this possibility, we
measured M-CSF in the supernatants of T-cell CM by ELISA. IL-7 stimulation of T cells resulted in M-CSF secretion of approximately 9 ng/mL per 106 cells in a 48-hour period, a concentration
that is physiologically relevant, as we have found that 10 ng/mL of
rhM-CSF is sufficient to support RANKL-mediated OC formation in
vitro (data not shown). No M-CSF secretion was detected in unstimulated
T cells. Stimulation of T cells by IL-1 To verify that the enhanced osteoclastogenic activity induced by IL-7
CM was due to the presence of secreted RANKL and M-CSF, we added the
RANKL inhibitor OPG or neutralizing antibody to M-CSF, into cultures of
monocytes treated with IL-7-stimulated T-cell CM. A high dose of OPG
(1 µg/mL), a concentration that completely inhibited OC formation
induced by 100 ng/mL rhRANKL (data not shown), was found to reduce OC
formation in IL-7-stimulated T-cell cultures by approximately 60%
(Figure 6). This confirms that
biologically active RANKL is secreted by T cells into the medium.
However, because OPG did not completely block the stimulation of OC
formation induced by T cells, even at concentrations up to 4 µg/mL
(data not shown), the data suggest that other unidentified factors
distinct from RANKL also contribute to the stimulation of OC formation. M-CSF neutralization failed to block either the RANKL-dependent or
RANKL-independent OC formation induced by IL-7-stimulated T-cell CM,
using concentrations of antibody found to completely inhibit OC
formation induced by rhRANKL and 500 ng/mL of rhM-CSF (a 50-fold higher
dose than the concentration of M-CSF measured in T-cell supernatants by
ELISA). This result suggests the presence of additional factors capable
of substituting for M-CSF. The identity of such factors remains to be
determined.
In this study, we have investigated the hypothesis that the
enhanced production of IL-1 and TNF Of considerable interest is the demonstration of substantial osteoclastogenic activity in IL-7-stimulated T-cell CM. Osteoclastogenesis has heretofore been thought to require physical contact between stromal cells and OC precursors of the hematopoietic lineage.23 However, the finding that T cells secrete soluble M-CSF and the confirmation that they secrete soluble RANKL4,18 provide a mechanism to explain how T cells can promote OC formation in the absence of cell-cell contact. This enables these lymphocytes not only to impact local sites of inflammation by means of membrane- bound factors but also to play a crucial role in the systemic bone loss characteristic of rheumatoid arthritis through the production of secreted factors. Previous reports suggest that, in the mouse, increased production of soluble RANKL is the major, if not the exclusive, mechanism by which inflammation induces bone loss in vivo.4 In contrast, the finding that saturating concentrations of OPG (4 µg/mL) did not completely inhibit the formation of OCs induced by IL-7-stimulated T-cell CM suggests that human T cells can secrete factors that can support OC formation independently of RANKL. Previous reports have also suggested that the osteoclastogenic effects of IL-7 are a result of its ability to promote B-lymphopoeisis.11 Our findings do not support this hypothesis, as the data demonstrate that IL-7 fails to induce osteoclastogenesis in the absence of T cells. Thus, the osteoclastogenic activity of IL-7 results from its ability to activate the T cells production of osteoclastogenic cytokines. However, we cannot exclude the possibility that effects of IL-7 on other cell populations may contribute to increase OC formation. For example, B220+ pre-B cells, (a population known to be induced by IL-7 treatment11), have recently been reported to be capable of differentiating into OC precursors. Thus, IL-7 may support osteoclastogenesis by redirecting pre-B cells into an OC lineage.14 However, because OC formation requires the action of osteoclastogenenic cytokines on the osteoclast precursors, our data suggest that T cells are crucial to this process as well. Our study measuring IL-7 production was conducted using concentrations
of IL-1 and TNF The demonstration that IL-1 and TNF The differentiation of OC precursors into mature OCs is reported to
require the coordinated effects of RANKL and M-CSF.7,22 M-CSF is also a critical enhancer of OC life span. Thus, stimuli that
enhance the production of M-CSF in the bone marrow are likely to
enhance OC formation and stimulate bone resorption. In accordance with
this hypothesis, we have previously reported that IL-1 and TNF On the basis of our data and those of others, we now propose a new
mechanism (Figure 7), which may account
for the accelerated bone loss observed in inflammatory conditions such
as rheumatoid arthritis and periodontitis.
Submitted February 8, 2000; accepted May 9, 2000.
Supported in part by grants from the National Institutes of Health (AG 13534 from the National Institute on Aging to R.P. and AR-99-001 to M.N.W. and AR46370 to L.R from the National Institute of Arthritis and Musculoskeletal and Skin Disease), and a grant from the Barnes-Jewish Hospital Research Foundation to M.N.W.
M.N.W. and S.C. contributed equally to this work.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Roberto Pacifici, Division of Bone and Mineral Diseases, Barnes-Jewish Hospital, North, 216 S Kingshighway Blvd, St Louis, MO 63110; e-mail: pacifici{at}imgate.wustl.edu.
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G. Toraldo, C. Roggia, W.-P. Qian, R. Pacifici, and M. N. Weitzmann IL-7 induces bone loss in vivo by induction of receptor activator of nuclear factor kappa B ligand and tumor necrosis factor alpha from T cells PNAS, January 7, 2003; 100(1): 125 - 130. [Abstract] [Full Text] [PDF]
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 A. Llano, J. Barretina, A. Gutierrez, J. Blanco, C. Cabrera, B. Clotet, and J. A. Este Interleukin-7 in Plasma Correlates with CD4 T-Cell Depletion and May Be Associated with Emergence of Syncytium-Inducing Variants in Human Immunodeficiency Virus Type 1-Positive Individuals J. Virol., November 1, 2001; 75(21): 10319 - 10325. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
) and osteoblasts
(
) (see "Materials and methods") were stimulated with IL-1 (1 ng/mL) and TNF
) were cultured
with or without rhIL-7 (2.5 ng/mL) or CM from purified T cells (
)
cultured in the presence or absence of rhIL-7. After 7 to 10 days,
TRAP+ multinucleated cells were counted. Only
IL-7-stimulated T-cell replete cultures and IL-7-stimulated T-cell CM
were able to stimulate appreciable OC formation. Data points are shown
as average + SD of 3 replicate wells. Data are representative of 3 independent experiments.
