Tumor Necrosis Factor Regulates Integrin Expression by Osteoclast ...

0013-7227/00/$03.00/0 Endocrinology Copyright © 2000 by The Endocrine Society

Vol. 141, No. 1 Printed in U.S.A.

Tumor Necrosis Factor a Regulates avb5 Integrin Expression by Osteoclast Precursors in Vitro and in Vivo* MASARU INOUE, F. PATRICK ROSS, JEANNE M. ERDMANN, YOUSEF ABU-AMER, SHI WEI, AND STEVEN L. TEITELBAUM Department of Pathology, Washington University School of Medicine, St. Louis, Missouri 63110 underscored by the capacity of an av inhibitory peptide mimetic to prevent spreading by BMMs expressing abundant avb5 while failing to impact those in which the integrin has been diminished by TNF. Finally, b5 mRNA in BMMs of wild-type mice administered lipopolysaccharide (LPS) progressively falls with time of in vivo treatment. Alternatively, b5 mRNA does not decline in BMMs of LPS-treated mice lacking both TNF receptors, documenting down-regulation of the b5 integrin subunit, in vivo, is mediated by TNF. Thus, matrix attachment of osteoclast precursors and mature osteoclasts are governed by distinct av integrins which are differentially regulated by specific cytokines. (Endocrinology 141: 284 –290, 2000)

ABSTRACT Early osteoclast precursors, in the form of murine bone marrow macrophages (BMMs), while expressing no detectable avb3 integrin, contain abundant avb5 and attach to matrix in an av integrin-dependent manner. Furthermore, avb5 expression by osteoclast precursors progressively falls as they assume the resorptive phenotype. We find the osteoclastogenic agent, tumor necrosis factor-a, (TNF) down-regulates avb5 expression by BMMS via attenuation of b5 messenger RNA (mRNA) t1/2. Using BMMs from TNF receptor knockout mice we establish the p55 receptor transmits the b5 suppressive effect. The functional implications of TNF-mediated avb5 down-regulation are

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ferentiate in culture (6). Moreover, cytokines and steroids that impact osteoclast differentiation (6) accelerate appearance of avb3 on osteoclast precursors and do so by regulating the b3 subunit (6, 7). While these observations are in keeping with the hypothesis that avb3 plays a pivotal role in the matrix degrading capacity of the mature polykaryon, they fail to address the means by which mononuclear osteoclast precursors recognize and attach to matrix. In this regard, we find avb3 undetectable on early, marrow-derived osteoclast precursors, despite their capacity to bind to extracellular matrix protein recognized by this integrin (6). This conundrum is a reflection of the fact these avb3 negative cells express the closely related integrin avb5, suggesting the latter heterodimer mediates osteoclast precursor-matrix recognition. In fact, as marrow macrophages assume the osteoclast phenotype in culture, avb5 disappears pari passu with emergence of avb3 (6). Given osteoclastogenesis is attended by diminution of avb5, one would expect agents that accelerate osteoclastogenesis to down-regulate the integrin. Tumor necrosis factor (TNF) is a proinflammatory cytokine with potent bone resorptive capacity, exerting its effect by enhancing osteoclast recruitment (8 –11). We find this cytokine hastens disappearance, on osteoclast precursors, of avb5, a reflection of accelerated degradation of b5 subunit mRNA. The TNF effect on the b5 integrin is mediated through the p55TNF receptor and, reflecting the in vitro situation, the TNF agonist lipopolysaccharide (LPS), when administered in vivo, rapidly diminishes marrow macrophage b5 mRNA.

STEOCLASTS are physiological polykaryons, and the principal if not exclusive resorptive cells of bone. They are derived from macrophage precursors residing in diverse tissues, principally marrow (1). While the precise means by which osteoclast precursors undergo differentiation are incompletely defined, attachment to matrix is probably essential. For example, macrophage polykaryons, including osteoclasts, fail to develop in suspension culture (Teitelbaum, S. L., unpublished observation) but form in abundance when in contact with matrix. Thus, the capacity of osteoclast precursors to recognize and attach to matrix is pivotal to generation of the resorptive phenotype. Integrins, which are major negotiators of cell matrixattachment, are transmembrane heterodimers consisting of noncovalently linked a and b subunits. Given the importance of matrix recognition to osteoclast recruitment and function, attention has turned to identifying integrins that mediate these events. These experiments establish avb3 as an integrin essential to osteoclast function. For example, antibody blockade of avb3 blunts bone resorption in vitro and in vivo (2– 4), and the b3 integrin knockout mouse develops osteosclerosis due to osteoclast failure (5). Given the abundance of avb3 on multinucleated osteoclasts, it seems likely the integrin participates in bone degradation by the mature cell. Consistent with the this posture, avb3 is progressively expressed as osteoclast precursors difReceived June 22, 1999. Address all correspondence and requests for reprints to: Steven L. Teitelbaum, M.D., Department of Pathology, Washington University School of Medicine, Barnes-Jewish Hospital North, 216 South Kingshighway, St. Louis, Missouri 63110. E-mail: teitelbs@medicine.wustl.edu. * Supported in part by NIH Grants DE-05413, AR-32788 (S.L.T.), AR-42404 (F.P.R.), and grants from the Barnes-Jewish Hospital Foundation and Monsanto (Y.A.)

Materials and Methods Reagents Unless specified, all reagents were obtained from Sigma (St. Louis, MO).

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TNF REGULATES avb5 INTEGRIN EXPRESSION Isolation and culture of osteoclast precursors These procedures have been reported in detail (1, 12). Briefly, marrow cells were obtained from 6-week-old male C3H or transgenic mice by flushing femora and tibiae with a-modified Eagle’s medium (a-MEM). The cells were cultured in a-MEM supplemented with 10% FBS in the presence of 500 U/ml stage 1 macrophage (M)-CSF (13) for 24 h. The nonadherent population was collected and mononuclear cells were isolated by Ficoll-Hypaque gradient centrifugation (500 3 g, 15 min). The marrow- macrophage enriched population was recovered by centrifugation (500 3 g, 7 min) and 5 3 106 cells plated in a-MEM supplemented with 10% FBS on 100-mm plates. The cells, maintained for 3– 4 days, were supplemented each day with 500 U/ml M-CSF yielding a pure population of M-CSF dependent, adherent osteoclast precursors. In selected studies, the same adherent osteoclast precursors, maintained in the presence of 500 U/ml M-CSF throughout, were treated, with time, with indicated doses of murine recombinant TNF (Genzyme, Cambridge, MA).

Osteoclast generation Nonadherent bone marrow macrophages and the murine stromal line ST2 were cultured at a ration of 10:1 (12). Cultures were fed every third day, at which time fresh steroids (10 nm 1,25 dihydroxyvitamin D3 and 100 nm dexamethasone) were added. Osteoclasts and their precursors were freed of ST2 cells by treatment with 0.1% bacterial collagenase, 0.1% BSA in a-MEM for 2 h at 37 C (11, 12).

Transgenic mice C3H/HeN male mice (Harlan Sprague Dawley, Inc., Indianapolis, IN) were used. Transgenic mice include: (a) mice in which the p55TNF receptor gene has been deleted (14) (provided by Dr. Warner Lesslauer, Hoffmann-La Roche, Basel, Switzerland); (b) those in which the p75TNF receptor gene has been deleted (15); (c) those in which both the p55 and p75TNF receptor genes have been deleted by interbreeding dominant negative p55TNF receptor knockout mice with their counterparts from p75TNF receptor knockout mice (provided by Dr. Mark Moore, Genentech, Inc., South San Francisco, CA), and (d) their respective wild-type mice.

Northern blot analysis Total cellular RNA was isolated with TRIzol (Life Technologies, Inc., Gaithersburg, MD) and 5 mg per lane electrophoresed in 0.9% agarose gel containing formaldehyde and transferred to Hybond N (Amersham Pharmacia Biotech, Arlington Heights, IL). The membrane was prehybridized with hybridization buffer [5 3 SSPE, 5 3 Denhardt’s solution, 50% formamide, 0.1% SDS, 1 3 Background Quencher (Tel-Test Inc., Friendswood, TX) for at least 2 h at 42 C. Northern analysis was performed using mouse b5 (16) and b3 (17) integrin complementary DNAs (cDNAs) cloned in our laboratory, or human av cDNA kindly provided Dr. Eric Brown (Washington University School of Medicine, St. Louis, MO), 32P labeled by the random primer method (Roche Molecular Biochemicals, Indianapolis, IN). After 16 h hybridization, membranes hybridized with b5, b3 or av cDNAs were washed as previously described (18). For reprobing, membranes were submerged in 0.1%SDS at 100 C. To normalize for RNA loading, blots were finally reprobed with an end-labeled oligonucleotide specific to 18S RNA (19). For determination of mRNA stability, cells in 100-mm dishes were cultured with or without 6.0 ng/ml TNF for 1 day. Actinomycin D (5 mg/ml) was added to all plates and total RNA isolated 0, 2, 4, and 6 h later. Thirty micrograms per lane of RNA was fractionated in agarose, 0.0 Northern analysis was performed using a b5 cDNA and the results quantified by densitometry.

Nuclear run-on transcription assay Adherent bone marrow macrophage were cultured for 24 h with or without TNF 6.0 ng/ml and washed twice with ice-cold PBS. Nuclear isolation and in vitro transcription were performed as previously described (18). After transcription the nuclei were harvested and RNA was isolated with TRIzol. Equal amounts of freshly transcribed RNA as determined by trichloroacetic acid (TCA)-precipitable counts were hybridized in hybridization buffer to denatured DNA (5 mg/slot b5 cDNA,

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b3 cDNA, vector DNA, and human G3PDH (CLONTECH Laboratories, Inc. San Diego, CA). After 36 h of hybridization, the membranes washed with 1 3 SSPE 0.1% SDS for 20 min at 50 C, 0.1 3 SSPE 0.1% SDS for 20 min at 60 C twice.

Immunoprecipitation Cells were washed with PBS and surface-labeled with 125I as described (18). Cells were lysed in buffer containing 2% Renex 30, 10 mm Tris pH 8.5, 150 mm NaCl, 1 mm CaCl2, 1 mm AEBSF and 0.02% NaN3. Each lysate, containing equal TCA-precipitable counts, was incubated with Gammabind (Amersham Pharmacia Biotech, Piscataway, NJ) and precleared again with Gammabind plus whole rabbit serum for b5 or class matched monoclonal antibody for b3. The precleared lysate was immunoprecipitated with a polyclonal rabbit anti-b5 integrin subunit antibody kindly provided by Dr. Louis Reichardt (University of California, San Francisco, CA) or hamster monoclonal anti-b3 integrin subunit antibody (PharMigen, San Diego, CA). The immunocomplex was bound to excess Gammabind. The precipitate was recovered by boiling the beads in electrophoresis sample buffer and subject to 7% SDS-PAGE under nonreducing conditions. The gels were dried and subject to autoradiography.

TNF assay TNF in medium and serum was measured by ELISA (Genzyme, Cambridge, MA).

Cell morphology Osteoclast precursors were cultured in 48-well plates in 500 U/ml M-CSF. At subconfluence, cells were treated with various combinations of a single dose of TNF, and daily additions of a small RGD peptide mimetic, SC56631 (10 mm) (Searle, Skokie, IL) known to block avb3 and avb5 (20). After 3 days the cells were fixed with 2% paraformaldehyde and photographed.

In vivo experiments Four- to six-week-old mice were injected, ip, with 1 mg of LPG. Marrow macrophages were isolated, with time, and cultured for 24 h as described above. The cells were lysed and total RNA probed with avb5 integrin cDNA. In some experiments TNF in tail vein blood was measured by ELISA.

Results TNFa specifically regulates b5 integrin mRNA

BMMs were treated in a concentration-dependent manner with TNF for 24 h and probed, by Northern analysis, with av, b3, and b5 cDNAs. As seen in Fig. 1, the quantity of b5 messenger RNA (mRNA) progressively falls with increasing TNF. Attenuation of b5 mRNA is detectable at cytokine concentrations as low as 1.8 ng/ml. In contrast to b5, TNF fails to impact either av or b3 mRNA at concentrations as high as 60 ng/ml. b5 mRNA declines as early as 4 h and nadir is reached 8 h after a single addition of 6.0/ml TNF-a (Fig. 2). TNF-a decreases b5 integrin mRNA stability

We next turned to the molecular mechanisms by which TNF regulates steady state b5 mRNA levels, asking if the cytokine impacts transcription or message stability. To assess transcription, nuclei were isolated from TNF treated and control BMMs and run on studies performed. As seen in Fig. 3, TNF fails to alter transcription of the b5 integrin gene. b5 mRNA stability was determined by exposing TNFtreated and virgin BMMs to the transcriptional inhibitor, actinomycin D and, using Northern analysis, assessing mes-

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TNF REGULATES avb5 INTEGRIN EXPRESSION

FIG. 1. TNF specifically regulates osteoclast precursor b5 integrin mRNA in a concentration-dependent manner. Osteoclast precursors were treated for 24 h with increasing concentrations of TNF. Total RNA was extracted and probed, by Northern analysis, with murine av, b3, and b5 integrin cDNAs. av and b3 autoradiograms were developed for 5 and 7 days, respectively, and b5 autoradiogram, overnight.

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FIG. 3. TNF fails to alter b5 integrin gene transcription. Osteoclast precursors were maintained 6 6.0 ng/ml TNF for 24 h. Nuclei were isolated and incubated with 32P-UTP. Equal cpm were hybridized to excess b3, G3PDH or b5 cDNAs or empty vector (pCR11). (Representative of three experiments.)

FIG. 2. TNF regulates osteoclast precursor b5 integrin mRNA in a time-dependent manner. Osteoclast precursors were maintained 6 6.0 ng/ml TNF. Cells were killed with time and total mRNA derived from TNF treated (1) and control (2) cells probed with a b5 cDNA.

sage levels with time. By densitometric quantitation, t 1/2 of b5 mRNA in control and TNF-a treated cells are 5.1 and 1.9 h, respectively (Fig. 4). TNF-a regulates b5 integrin subunit mRNA via the 55-kDA TNF receptor

To identify the receptor mediating TNF modulation of b5 mRNA we took advantage of three strains of transgenic mice in which either the p55, p75 or both TNF receptors (TNFrs) are deleted. b5 mRNA declines in BMMs derived from wildtype mice challenged with TNF, whereas the cytokine fails to exert this effect on cells of animals lacking both TNFrs (Fig. 5). Thus, TNF modulates b5 integrin expression via a classical TNFr(s). Confirming that soluble TNF-induced b5 downregulation is mediated via the p55TNFr, BMMs of animals lacking this receptor, like those of double receptor knockout mice, fail to normally alter integrin mRNA levels in response to the cytokine. In contrast, p75TNFr2/2 mutant cells behave substantially like wild-type. TNF-a specifically decreases avb5 while not effecting avb3 expression

To determine if TNF reduction of b5 mRNA is reflected by surface expression of integrin heterodimers, BMMs were surface labeled with 125I, lysed, and the lysate immunopre-

FIG. 4. TNF accelerates b5 mRNA degradation. Osteoclast precursors were maintained 6 6.0 ng/ml TNF for 24 h. Total RNA was isolated before (0) and 2, 4, and 6 h following addition of Actinomycin D (5 mg/ml). Northern analysis was performed with a b5 cDNA and the autoradiograms densitometrically assessed. The data are presented as % b5 mRNA present at time 0. No detectable signal was obtained from mRNA derived from BMMs treated for 6 h with TNF.

cipitated with anti-b5 or b3 antibodies. As seen in Fig. 6, avb5 is expressed by these mononuclear osteoclast precursors while, by this technique, avb3 is virtually undetectable. Moreover, exposure of these cells to TNF (6.0 ng/ml) for 48 h diminishes avb5 abundance while failing to impact avb3. The TNF-induced decline in avb5 expression is accompanied by distinct morphological changes in adherent BMMs plated in serum containing medium, rich in vitronectin (Fig. 7). Thus, untreated cells assume a spindle shape, whereas those exposed to the cytokine appear more polygonal. Furthermore, a blocking peptide mimetic recognizing avb3 and avb5 (20) prevents spreading of virgin cells while not altering the morphology of TNF-exposed BMMs lacking both integrins. The latter observation indicates spreading of early osteoclast precursors is mediated by avb5, a phenomenon lost with TNF treatment.


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FIG. 5. TNF regulates b5 integrin mRNA via the 55 kDa TNF receptor. Osteoclast precursors derived from marrow of wild-type mice [p55(1)p75(1)] and those in which the p55 [p55(2)p75(1)] or p75 [p55(1)p75(2)] TNFr or both TNFrs [p55(2)p75(2)] are deleted were maintained for 8 h in increasing amounts of TNF. Total RNA was probed with a b5 integrin cDNA.

nadir reached at 2 h, after which b5 mRNA levels begin to increase (Fig. 10). Suggesting LPS suppression of b5 mRNA is negotiated by TNF, circulating levels of the cytokine measured 30 min earlier are inversely proportional to integrin message. To confirm down-regulation of b5 mRNA, in vivo, is mediated by TNF we isolated BMMs from endotoxintreated wild-type mice and those deleted of both the p55 and p75 TNFrs. Figure 11, and densitometric analysis of the data contained therein, reveal b5 message falls to 7.4% of control only in cells obtained from LPS-treated mice in which TNFrs are intact. In contrast, b5 mRNA in BMMs deleted of both TNFrs does not decline under the influence of endotoxin (109.3% of control). Furthermore, despite the abundance of b5 mRNA in wild-type compared with mutant virgin BMMs, the message is least in cells derived from LPS-treated wildtype mice. FIG. 6. TNF decreases avb5 but does not effect avb3. Osteoclast precursors, maintained 48 h 6 6.0 ng/ml TNF, were surface labeled with 125 I and immunoprecipitated with anti-b3 or b5 integrin antibodies. The immuno-precipitate was subjected to SDS-PAGE in nonreducing conditions and the gels autoradiographed.

TNF mRNA is expressed during osteoclastogenesis

To determine if TNF suppression of avb5 expression is biologically relevant, we asked if the cytokine is expressed during basal osteoclastogenesis. To this end, osteoclastogenic cultures consisting of BMMs and ST2 stromal cells were established. The stromal cells were removed, with time, and the remaining cells, consisting entirely of mononuclear precursors or mature osteoclasts (11, 12), probed for TNF mRNA. As seen in Fig. 8, freshly isolated (day 0) BMMs lack detectable TNF message, which is abundantly expressed within the first day of osteoclastogenic culture. Given the fact osteoclasts emerge in this system on day 6 –7 and progressively increase with time, TNF mRNA levels are ample in osteoclast precursors but incrementally decline with appearance of the mature resorptive polykaryon. LPS induces TNF expression by BMMs and decreases b5 mRNA levels ex vivo

LPS is a potent inducer of TNF and such is the case regarding BMMs (11). While TNF in medium conditioned for 8 h by virgin BMMs is undetectable, the cytokine is abundant in that containing cells exposed to LPS (Fig. 9). Furthermore, b5 mRNA content of BMMs isolated, with time, from LPS injected animals, and cultured for 48 h in LPS free conditions, falls with duration of LPS exposure in vivo. The effect is apparent in cells exposed to LPS for 20 min in vivo with the

Discussion

Bone resorption is reflective of the rate at which osteoclasts degrade bone and the efficiency of osteoclast precursor differentiation. The bone degradative process by the mature polykaryon depends upon creation, at the cell-bone interface, of a highly acidic, isolated microenvironment (21). The gradient between the pH of the general extracellular space and that extant where the osteoclast contacts bone indicates physical intimacy between cell and matrix is fundamental to the resorptive process, an event involving the integrin, avb3 (2– 4). Cell-matrix recognition is also essential to osteoclast differentiation but the means by which mononuclear precursors recognize substrate are unknown. We find osteoclast precursors, in the form of isolated marrow macrophages, contain no detectable b5 mRNA or avb5 (6). These immature cells can, however, attach to and spread on vitronectin (6). This observation prompted us to search for a related heterodimer, expressed by early osteoclast precursors, which also binds this avb3 ligand. In fact, these immature cells contain an abundance of avb5, an integrin structurally similar to avb3 also recognizing the RGD amino acid sequence resident in a number of matrix proteins including osteopontin, bone sialoprotein and vitronectin (22–24). Osteoclast precursors, as they differentiate into mature resorptive polykaryons, however, lose avb5 and, in a reciprocal fashion, express avb3 (6). Taken together, these findings suggest matrix recognition and attachment by early osteoclast precursors is mediated by avb5 whereas the role of avb3 is confined to more committed cells. avb5, while structurally related to avb3, appears to mediate distinct events such as uptake of vitronectin, facilitated entry of adenovirus type 2 and enhanced angiogenesis (25). Our

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FIG. 7. An av integrin antagonist alters the morphology of virgin but not TNF-treated osteoclast precursors. Osteoclast precursors were maintained 72 h with or without 6.0 ng/ml TNF and/or the anti avb3-avb5 peptide mimetic SC56631. The cells were visualized by phase contrast microscopy. (2 and 1 refer to absence or presence, respectively, of TNF or/56631).

FIG. 8. TNF mRNA is expressed early in osteoclast differentiation. Osteoclastogenic cultures consisting of a pure population of marrow derived osteoclast precursors to which the ST2 murine marrow stromal line had been added, were established. Stromal cells were removed by collagenase digestion, with time, and residual total RNA derived from osteoclast precursors or mature osteoclasts, probed with a murine TNF cDNA. Day 0 represents pure population of osteoclast precursors before addition of ST2 cells.

finding that avb5, and not avb3, mediates osteoclast precursor spreading in an RGD dependent manner also indicates the two integrins functionally differ. TNF, among the most potent of resorptive cytokines, dramatically increases osteoclastogenesis in man (10) and mouse (11) marrow culture. Given its effect on osteoclast differentiation, we asked if TNF also impacts av integrin expression by isolated osteoclast precursors. avb5 is indeed down-regulated on early osteoclast precursors exposed to the cytokine. This event is paralleled by changes in b5 mRNA reflecting message destabilization and not attenuated transcription.

Because TNF, in other circumstances, stabilizes macrophage mRNA (26) its impact on b5 message is not a reflection of nonspecific, accelerated degradation. In contrast to b5, av message remains unaltered by TNF. The fact b5 associates only with av, whereas av partners with a number of b subunits, indicates it is the monogamous b, and not promiscuous a chain, which regulates expression of the heterodimer. This finding is in keeping with the capacity of cytokines such as IL-4 (18) and steroids such as retinoic acid (7) to modulate avb3 via the b3 subunit. Precisely why avb5, abundant on osteoclast precursors,


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FIG. 11. LPS inhibition of b5 mRNA expression, in vivo, is mediated by TNF. LPS was administered to wild-type [p55(1)p75(1)] or double TNFr deleted [p55(2)p75(2)] mice. The animals were killed after 3 h and osteoclast precursor total mRNA probed with a b5 cDNA FIG. 9. LPS induces TNF secretion by osteoclast precursors. Osteoclast precursors (4 3 105/well) were maintained for 8 h in 24-well plates, 6 100 ng/ml LPS. TNF concentration in conditioned medium was determined by ELISA.

FIG. 10. LPS, in vivo, decreases b5 mRNA. Top panel, LPS injected C3H mice were killed, with time, and osteoclast precursor total RNA probed with a b5 cDNA. Bottom panel, Circulating TNF levels were measured, by ELISA, before and at various times after LPS administration.

disappears as the cells differentiate into resorptive polykaryons is unknown. It is of interest, however, that avb5, in contrast to the mature osteoclast integrin avb3, mediates cell spreading. In fact, TNF, by blunting avb5 expression, diminishes the capacity of osteoclast precursors to spread. This observation is in keeping with reports that osteoclasts actively resorbing bone fail to assume the spread configuration observed in their inactive counterparts (27). Thus, resorption may require intermittent attachment of osteoclasts to bone with motility inhibited by cell spreading.

The p55TNFr has been viewed as the major transmitter of TNF-induced signals although recent evidence indicates both p55TNFr and p75TNFr are functional (28). For example, while p75TNFr mediates differentiation of early hematopoietic precursors, p55TNFr is active in late stages of the process. On the other hand, soluble, compared with membraneresiding TNF, targets primarily, if not exclusively, p55TNFr (29), through which it promotes osteoclastogenesis (11). To determine if down-regulation of avb5 on BMMs, an event characterizing commitment of these cells to the osteoclast phenotype (6), is also mediated by p55TNFr, we turned to mice lacking combinations of the two TNFrs. Confirming the TNF-induced fall in b5 is negotiated via a classical TNFr, osteoclast precursors derived from double receptor deficient mice fail to alter expression of the integrin in response to the cytokine. Most importantly, failure of p55 but not p75 TNFr2/2 BMMs to meaningfully dampen b5 mRNA in response to the soluble cytokine establishes the p55 species as the principal mediator of the integrin suppressive signal. Perhaps our most compelling evidence supporting the biological significance of TNF-induced avb5 down-regulation are our experiments involving LPS. We find LPS-exposed osteoclast precursors, like other macrophages (30 –32), secrete levels of TNF comparable to those used in our in vitro experiments. Importantly, administration of this TNF agonist, to mice, dampens b5 expression by osteoclast precursors, ex vivo. Interestingly, the time course of b5 regulation by in vivo LPS mirrors commitment of BMMs to the osteoclast phenotype (11). Mice devoid of both TNFrs fail to downregulate b5 mRNA when administered LPS establishing the phenomenon is mediated by TNF. The rapid suppression of b5 in the in vivo state may be reflective of the complex manner by which LPS induces expression of the cytokine (33). These events, involving transcriptional and posttranscriptional phenomena, are known to promote TNF secretion by macrophages within minutes of endotoxin exposure. Our experiments, particularly those performed in vivo, suggest TNFmediated b5 down-regulation may obtain in pathological states such as bacterial infection which, as a component of periodontal disease, prompts profound bone loss.


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