Tumor necrosis factor-α mediates polymethylmethacrylate particle-induced NF-κB activation in osteoclast precursor cells

ELSEVIER

Journal of Orthopaedic Research

Journal of Orthopaedic Research 20 (2002) l7&181

www.elsevier.com/locate/orthres

Tumor necrosis factor-a mediates polymethylmethacrylate particle-induced NF-tiB activation in osteoclast precursor cells John C. Clohisy a,*, Steven Teitelbaum ', Shaoping Chen Yousef Abu-Amer

a,

Jeanne M. Erdmann

a,

Department of Orthopaedic Surgery. Barnes-Jewish Hospital at W'ashington Unirersity School of' Medicine, One Barnes-Jewish Hospital Plaza, Suite 11300, West Puirilion. St. Louis. M O 63110, USA Depurtrnent of Pathology. Washington Chiaersity 3 h o o l of Medicine, St. Louis, M O 63110, U S A

Abstract

Tumor necrosis factor-sc (TNF) is a potent osteoclastogenic cytokine that has a fundamental role in the pathogenesis of implant particle-induced osteolysis. The nuclear transcription factor NF-KB mediates TNF signaling and this transcription complex is necessary for osteoclastogenesis. Because polymethylmethacrylate (PMMA) particles cause osteolysis, we reasoned the PMMA would induce NF-tiB activation. In fact, we find that exposure of osteoclast precursors, in the form of colony stimulating factor-1 (CSF-1)dependent murine bone marrow macrophages, to PMMA particles prompts nuclear translocation and activation of NF-KB. Supershift assays confirm the presence of the p50 and p65 NF-tiB subunits in the activated transcription factor. Particle-induced NF-tiB activation is equal in both wild type and LPS- hyporesponsive cells indicating that the phenomenon does not represent endotoxin contamination. A soluble, competitive inhibitor of TN F (huTNFr:Fc) dampens particle-directed NF-h-B activation and this response is also abrogated in TNF-'- osteoclast precursors. Thus, PMMA particle activation of NF-KB is a secondary event resulting from enhanced TNF expression and is independent of LPS contamination. 0 1002 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved.

Introduction

Particulate-induced periprosthetic osteolysis is the major cause of prosthetic joint implant loosening [l 1,16,19]. This implant-associated bone loss results from a chronic granulomatous inflammatory response that is elicited and sustained by implant-derived particles. Polymethylmethacrylate (PMMA), polyethylene and metal debris enhance local secretion of proinflammatory factors including interleukin- 1, interleukin-6, TNF and prostaglandin E2 [10,12,15,16] which mediate differentiation of osteoclast precursor cells into bone resorbing mature osteoclasts. The exact mechanism of action and the specific role of each proinflammatory factor in this pathologic response are unclear, yet T N F has emerged as perhaps, the critical cytokine [ 17,25,26]. This potent osteoclastogenic factor is present in periprosthetic membrane tissue obtained from loose im-

*Corresponding author. Tel.: +I-314-417-2566; fax: +1-314-7472599. E-mail nddress: jclohisyc~~msnotes.wustl.edu (J.C. Clohisy).

plants [7], and macrophage expression of T N F in vitro is increased by exposure to implant-associated particles [6,7,10,12,13,15-171. Additionally, genetic and pharmacological blockade of T N F signaling prevents PMMA particle-induced osteolysis [ 17~5,261.These findings point toward the T N F signaling pathway as playing a major role in implant particle-stimulated bone loss. In many cell types, the nuclear transcription factor NF-KB mediates the gene regulatory function of TNF. Members of the NF-riB family form functional diniers that act as inducible transcription complexes and regulate target cell gene transcription [2,4,8]. Classically, the inactive NF-tiB heterodimer is located in the cytoplasm in a high-affinity association with a labile cytoplasmic inhibitor protein, ItiB. Most commonly, NF-tiB is activated secondary to phosphorylation of ItiB. Upon ItiB phosphorylation, NF-tiB is released, translocates to the nucleus and binds to specific DNA response elements where it exerts its transcription regulatory function. NFKB regulates gene expression in a variety of biological responses [4,28] and is essential for developmental osteoclastogenesis. In fact, mice deleted of the p50/p53 NF-KB heterodimer develop osteopetrosis in which the

0736-0266/02/$ - see front matter 0 2001 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved PII: S 0 7 3 6 - 0 2 6 6 (01 ) 0 0 0 8 8 - 2

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animals lack osteoclasts [3,14]. Thus, we hypothesized that implant debris activates NF-tiB, in osteoclast precursor cells, via the T N F signaling pathway, and that this NF-tiB activation is fundamental to particleinduced osteoclastogenesis. In this study, we have explored the effect of PMMA particles on NF-tiB activity in osteoclast precursor cells (CSF-1 dependent bone marrow macrophages) and its relationship to TNF. We find that PMMA particles activate NF-KB in authentic osteoclast precursors as a secondary event to T N F stimulation and do so in an LPS-independent manner. Most importantly, genetic and pharmacological blockade of T N F obviates particle-induced NF-tiB activation. Thus, arrest of T N F or NF-tiB activity present themselves as possible therapeutic targets in implant osteolysis.

175

irregular shapes and smooth surfaces. The majority of these submicron particles were in aggregates and are in the biologically active size range when compared to particulates isolated from periprosthetic membrane tissue [16-181. To test for particle Contamination with LPS, a commercially available technique (E-toxate Kit) was employed. No LPS was detected by this assay in this PMMA particle preparation. Imniunohlorring

Methods

Nuclear or cytoplasmic lysates were isolated and then boiled in sample buffer (0.5 M Tris-HCI pH 6.8, lo'!,%(wlv) SDS, 10'1.;) glycerol, 0.05'%,(w/v) bromophenol blue, distilled water) for 5 min and proteins separated by gel electrophoresis (8-12% gradient SDS-PAGE). Proteins were then transferred to nitrocellulose membrane by a semi-dry blotter (Bio-Rad, Richmond, CA) and incubated in blocking solution (lo'%,skim milk, 0.05% Tween-20 in PBS). Nitrocellulose membranes were then washed with PBSlTween buffer and exposed to the primary antibodies for 1 h at room temperature. After the primary antibody incubation, the membranes were washed four times and incubated with the appropriate horse radish peroxidase-conjugated secondary antibody for 1 h at room temperature. The membranes were then washed extensively (5 15 min) and subjected to an enhanced chemiluminescence detection assay according to the manufacturer's protocol. Immunoblot results were confirmed in three independent experiments with similar results.

Reuxmts

Electrophori~ticMobility Shifi Assuy ( EMSA J

Recombinant murine TNF was purchased from R&D Systems (Minneapolis, MN). Polyclonal anti-p5ONF-tiB, antLp52NF-tiB and anti-p65NF-~Bantibodies were purchased from Santa Cruz (Santa Cruz, CA). The E-toxate kit was obtained from Sigma (St. Louis, MO) and huTNFr:Fc (Enbrel) was supplied by Immunex (Seattle, WA).

Isolation of nuclear fractions was performed as previously described [27]. In short, control or particle-stimulated monolayers of osteoclast precursor cells grown in 100 mm' tissue culture dishes were washed twice with ice-cold phosphate-buffered saline. Cells were lifted from the dish by treating with 5 mM EDTA and 5 mM EGTA in PBS. Cells were then resuspended in hypotonic lysis buffer A (10 mM HEPES [pH 7.81, 10 niM KCI. 1.5 m M MgCl,, 0.5 mM dithiothreitol, 0.5 mM AEBSF, 5 (pglml Leupeptin), incubated on ice for 15 min, and Nuclei were pelleted NP-40 added to a final concentration of 0.64'%1. and the cytosolic fraction carefully removed. The nuclei were then resuspended in nuclear extraction buffer B (20 mM HEPES [pH 7.81, 420 mM NaCI, 1.2 mM MgCI,, 0.2 mM EDTA 25"h glycerol, 0.5 mM dithiothreitol, 0.5 mM AEBSF, 5 (pglml Pepstatin A), 5 (pg/ml Leupeptin), vortexed for 30 s and rotated for 30 min at 40°C. The samples were then centrifuged and the nuclear proteins in the supernatant transferred to fresh tubes and protein content measured using a standard bicinchoninic acid protein assay. Nuclear extracts (I0 pg) were incubated with an end-labeled double stranded oligonucleotide probe containing the sequence 5'- AAA CAG G G G GCT TTC CCT CCT C -3' [9] derived from the riB3 site of the T N F promoter. The reactions were performed in a total of 20 pl of binding buffer (20 mM HEPES [pH 7.81, 100 mM NaCI, 0.5 mM dithiothreitol, I pg poly dIL dC, and 10'!41glycerol) for 15-20 min at room temperature. After incubation with the labeled probe for an additional 30 min, samples were fractionated on a 40'0 polyacrylamide gel and visualized by exposing the dried gel to autoradiographic film. For supershift assays the antibody to the NF-KB subunit of interest was added to the binding reaction. EMSA findings were confirmed in at least three independent experiments with similar results.

Cell culiurelniiw

Osteoclast precursor cells, in the form of murine bone marrow macrophages, were isolated from whole bone marrow as previously described [1,2,8]. In brief, marrow cells of 4-6 week-old mice [C3H/ HeN or C3HIHeJ (Harlan Industries, IN) or TNF-'- mice [20]] were flushed from freshly harvested femurs and tibias and then incubated in tissue culture plates, at 37°C in 5% COz. in the presence of CSF-I (10 nglml). In order to separate the contaminating stromal cells (initially adherent) from the bone marrow macrophage precursors (initially non-adherent), after 24 h in culture the initially adherent stromal cells were discarded. The non-adherent cells were isolated and layered on a Ficoll-Hypaque gradient. The cells at the gradient interface (95% enriched inonocytelmacrophage population) were collected. plated in s ~ MEM (supplemented with 10%,heat-inactivated fetal bovine serum) at 37°C in 5% C 0 2 in the presence of CSF-1 ( I 0 nglml). This non-stromal cell population in the presence of CSF-I subsequently becomes adherent and represents a relatively pure population of bone marrow macrophages. This is demonstrated by the facts that 99% of seven days old colonies are positive for monocyte-specific a-napthyl acetate transferase activity and 99% of these cells die within 72 h of CSF-I removal [8]. These CSF-1 dependent bone marrow macrophages (osteoclast precursors) were then treated according to the desired experimental conditions. Polymethylnzcthucr~lutepur ticles Commercially available, gamma radiation-sterilized PMMA particles (Howmedica, Simplex-P) were used for all experiments. The powder component (mixture of PMMA, methyl methacrylate-styrenecopolymer and barium sulfate) of the two component (methyl methacrylate mixture and liquid monomer) bone cement preparation was used as provided in the commercial product without modification. Stock solutions were prepared with a-MEM at a concentration of 100 mg/ml and treatment concentrations are indicated for each experiment. Scanning electron microscopy revealed a heterogeneous array of particle sizes (range, 220 nm to 60 pm in diameter) and shapes. Less than 5% of the particles consisted of relatively large smooth-surfaced microspheres in the 20-60 pm range. The majority (at least 50%) of the particles were smaller in the 220 nm to 1 pm range and had slightly

Results P M M A particles artiiute NF-tiB We have previously shown, in an experimental murine osteolytic model, that blockade of the T N F signaling pathway prevents PMMA particle-induced osteolysis [17]. This observation, taken with the fact that NF-KB mediates most TNF-induced events, prompted us to ask if the transcription factor also participates in this pathologic process. We first examined the effect of

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PMMA particles on NF-tiB activity in murine osteoclast precursors. Thus, using antibodies to the p50, p52 and p65 NF-tiB subunits, we performed immunoblot analysis of cytosolic (Fig. l a) and nuclear (Fig. l b) fractions isolated from untreated bone marrow macrophages and those exposed, with time, to PMMA particles. The p50 NF-tiB subunits increased in both the cytosolic and nuclear fractions after exposure to particles, while the p65 subunit decreased in the cytosol(70% decrease at 40 min) and was enhanced in the nuclear fraction indicat-

Treatment Time (min.)

c

Lps

0

20

PMMA

20

40

60

P65

P50

Treatment Time (min.)

C

PMMA

m 0

20

40

60

P65

P50 (b)

Fig. 1. PMMA particle exposure stimulates nuclear translocation of the p50 and p65 NF-rtB subunits. Cytosolic (a) and nuclear (b) protein extracts were isolated from osteoclast precursors after treatment with the control media, PMMA (0.75 mg/ml) or LPS (5 ng/ml) for the times indicated. Equal amounts of extract protein were electrophoresed, transferred to nitrocellulose membrane and analyzed by inimunoblotting with anti-p50 and anti-p65 antibodies.

ing translocation to the nucleus. Furthermore, attesting to its activity, NF-tiB, translocated to the nucleus following particle exposure binds DNA, in a time (Fig. 2a) and particle dose-dependent manner (Fig. 2b), as determined in electrophoretic mobility shift assays, using the tiB3 sequence of the T N F promoter as a probe. Enhanced tiB3 binding activity is detected at 15 min, maximizes at 2 h and returns to control levels by 24 h. A relatively narrow dose-response range was identified with an optimal response at a concentration of 0.75 mg/ ml. As expected, the positive controls LPS ( 5 ng/ml) and T N F (5 ng/ml), also increases tiB3 binding activity within 15 min after treatment and at optimal concentration these factors (LPS and TNF) are more potent activators of NF-tiB when compared to the PMMA particles. This may be due to rapid primary responses with T N F and LPS compared to the delayed secondary PMMA response. The specificity of this EMSA for NFKB activity was tested by repeating the experiment with 50 fold excess of non-radiolabeled tiB3 oligonucleotide or of a mutant oligonucleotide (Fig. 2c). Establishing specificity of the observed DNA binding, the nonradiolabeled tiB3 probe abolishes the event, while a mutant oligonucleotide only slightly decreases the binding activity. To confirm the results of our immunoblot experiments that demonstrated p50 and p65 NF-tiB subunit nuclear translocation indicating these subunits as components of the PMMA-activated dimer, we performed supershift assays. The p52 NF-tiB subunit was also tested because of its established role in developmental osteoclastogenesis [ 141. Nuclear extracts from PMMA treated and control bone marrow macrophages were tested with antibodies to the p50, p52 and p65 NFKB subunits. The addition of antibody to the p50 (Fig. 3a) or p65 (Fig. 3b) subunits evokes a slower migrating supershift band confirming the presence of these subunits in the activated NF-tiB dimer. Antibody to the p52 subunit did not elicit a supershift band nor have a major effect on the intensity of the PMMA-induced NFtiB band shift. These experiments are consistent with the hypothesis that the p50 and p65 NF-tiB subunits are mobilized to the nucleus as components of PMMAactivated NF-tiB. The results do not provide definitive evidence that the p52 subunit is mobilized, yet it is possible that this subunit is involved to some extent. P M M A actiimtion of NF-KB d0e.Y con tamination

lint

reject LPS

LPS, a known activator of NF-tiB in various cell types including osteoclast precursors and a potent stimulator of osteoclastogenesis [l], has recently been implicated in periprosthetic osteolysis [5,23]. Thus we analyzed the effect of LPS on NF-tiB activity in C3H/ HeN murine osteoclast precursors. LPS stimulates a time (Fig. 4) dependent increase in NF-KB activity with

J. C. C'lohisy et al. I Journal of Orthopedic Research 20 (2002i 17.1-181

Treatment Time

PMMA

TNF LPS Y n r ?

,/

C 15m 30 60 90

177

2h 4

6 15m 15m

Treatment Time(rnin.) ~I

I PS

PMMA (Im#:mli

PMMA(0 75mgirnlJ

P h N A (U 5mgiml)

n 0 30

20 40 GO I20 20 40 60120 20 40 GO 120

NFKB

NFKB

(c)

Fig. 2. PMMA Particles Activate NF-KB in Osteoclast Precursor Cells. ( a ) Osteoclast precursor cells were treated with control media or media containing PMMA particles (0.75mglml) for the times indicated. LPS (5 ng/ml) and TNF (5 ng/ml) served as positive controls. Cells were harvested and nuclear extracts were prepared for electrophoretic mobility shift assay (EMSA). Binding reactions were performed with the double stranded ~ site of the TNF prooligonucleotide probe containing the sequence 5AAA CAG G G G GCT TTC CCT CCT C-3derived from the K B (NF-KB) moter. The activated NF-KBband shift is indicated and the free unbound nucleotide probe is at the bottom of the autoradiogram. (b) Osteoclast precursors prepared were treated with various PMMA particle concentrations for the indicated time intervals and EMSA of the nuclear extracts was performed with the tiB3 probe. LPS (5ng/ml) was the positive control. (c) EMSA specificity was confirmed by performing the nuclear extract binding reactions (extracts form control or PMMA-treated cells) in the absence or presence of 50 fold excess unlabeled tiB3 or a mutated oligonucleotide probe, as indicated.

enhanced activity detected 5 min after treatment and a maximal respoiise between 30 and 120 min. To determine if PMMA-induced activation of NF-KB is the result of particle-associated LPS, osteoclast precursors from control (C3HeN) and LPS-hyporesponsive (C3Hl HeJ) mice [22] were treated with PMMA (2 mg/ml) or an optimal dose of LPS ( 5 ng/ml). As demonstrated by EMSA (Fig. 4b), PMMA and LPS enhance tiB3 binding activity in wild type osteoclast precursors. However, in osteoclast precursors from LPS-hyporesponsive mice, LPS-induced NF-tiB activation is abolished, while the

PMMA effect remains unchanged. Thus PMMA particle activation of NF-tiB is not due to LPS contamination.

TNF mediutes PMMA-induced NF-KBucthution Particulate debris stimulates expression of several proinflammatory factors [ 161 in osteoclast precursors which through autocrine mechanisms could invoke the observed PMMA-induced NF-tiB activation. If TNF plays a significant role in this response, blockade of the T N F signaling pathway should attenuate the PMMA

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Treatment

c

Time

0 15m 30 5m15 30 60 90 1204h 6h

TNF lOng/ml 1

LPS lOng/ml

Y

Supershift NFKB

NF KB

N v r

v r w a R

Supershift

NFKB

NFKB

(b)

(b)

Fig. 3. PMMA particle-activated NF-tiB dimer contains the p50 and p65 subunits. Nuclear extracts were prepared from osteoclast precursors after 2 h of exposure to control media or media containing PMMA particles (0.75 mgiml). Supershift assays were performed with antibodies to the p50 (a), p51 or p65 (b) NF-tiB subunits. The K B ~ oligonucleotide probe was used. NF-KB and supershift bands are marked.

effect. To address this issue, we turned to a competitive inhibitor of TNF, namely the soluble p75 T N F receptor external domain expressed as an Fc fusion protein (huTNF:Fc).Pretreatment ( 1 h) of the cells with huTNF:Fc, alone, does not effect basal NF-tiB activity, yet PMMA particle-induced NF-KB activation is abrogated (Fig. 5). To further support the role of T N F as a mediator of PMMA-induced NF-KB we compared

Fig. 4. Polymethylmethacrylate particles activate NF-tiB independent of LPS. Osteoclast precursor cells (C?H/HeN)were treated with control media, media containing LPS (10 ng/ml) or TNF (10 ng/ml) and harvested at the times indicated ( a )to determine the effect of LPS over time. Nuclear extracts were isolated for EMSA analysis with the tiB3 oligonucleotide probe. The NF-tiB band shift is identified and free oligonucleotide probe is present at the end of the gel. Osteoclast precursor cells were then isolated from control mice (C3HIHeN) or LPSresistant mice (C3HIHeJ) and treated with control media, PMMA particles (1.0 mglml) or LPS (5 nglml). After 2 h, cells were harvested and nuclei prepared for EMSA (b).

NF-tiB activation, by PMMA (0.75 mg/ml), T N F ( 5 ng/ ml) and LPS (5 ng/ml) in osteoclast precursors isolated from control mice (C3H/HeN) to those deleted of the T N F gene (Fig. 6). Contrary to significant timedependent activation of NF-KB in wild type mice, particle activation of NF-tiB in TNFPi4 cells is markedly reduced. These results suggest that T N F is required to mediate the secondary PMMA activation of NF-iiB.

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of' Orthopedic Rescrrrch 20

NFKB

Fig. 5. Soluble p75 T N F Receptor Blocks Particle-Induced NF-KB Activation in Osteoclast Precursor Cells. After three days of maintenance culture, osteoclast precursor cells were treated for 1 h with control media, TNF (5 ng/ml), PMMA particles (0.75 mg/ml, 1.0 mg/ ml) huTNF:Fc (1.5 pg/ml), or the indicated combinations of treatments. Nuclear extracts were analyzed by EMSA using the tiB3 consensus sequence probe for binding reactions. The NF-tiB band is marked and free probe present at the bottom of the autoradiogram.

TNF(-/-)

WT Treatment Time(min.)'O

PMMA 15 30 60

PMMA

'INF LPS

1 2 3 30 30

O/

15 30 60

rNF LPS 120' 30 30

NFKB

Fig. 6 . PMMA particle activation of NF-KB is abolished in TNF-'osteoclast precursors. Osteoclast precursor cells isolated from control mice (C3HIHeN) or from TNF-/- mice were exposed to the indicated concentrations of PMMA (0.75 mg/ml), TNF (5 mg/ml) or LPS (5 ng/ ml). Cells were harvested and nuclear extracts prepared for EMSA. The NF-rcB band shift is labeled and free probe is seen at bottom of autoradiogram.

Discussion Various osteoclastogenic factors, including TNF, IL-1, IL-6 and PGE1, may be involved in the patho-

(2002) 17.1-181

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genesis of particle-stimulated osteolysis [ 161. Identification of the specific cytokine mediators of this form of pathological bone loss, and delineating the responsible signaling pathways, may ultimately lead to the development of new molecular targets for therapeutic intervention [24]. Our previous work suggests that TN F has a fundamental role in implant osteolysis, thus prompting us to focus on this cytokine in elucidating the events that result in particle-associated bone loss. The nuclear transcription factor NF-tiB is a known mediator of TNF-induced gene transcription and is also essential for developmental osteoclastogenesis [3,14]. We, therefore, asked if NF-tiB is activated by particulate debris and, if T N F plays a role. We show that PMMA particles activate NF-tiB in mouse osteoclast precursors (CSF-1 dependent bone marrow macrophages) as a secondary event to T N F stimulation. Taken with previous reports that titaniumalloy particles activate NF-tiB in murine splenocytes [25] and human macrophages [18] our observations, and the absence of osteoclasts in the NF-tiB deleted mouse [14], implicate this transcription factor in particle-induced osteoclastogenesis. Since T N F is fundamental to experimental implant osteolysis, we investigated the role of this cytokine in the observed particle-directed activation of NF-KB. Blockade of the T N F pathway with huTNF:Fc, which binds and sequesters TNF, abolishes PMMA activation of NF-tiB and the transcription complex is not mobilized in particle-treated TNF-/osteoclast precursors. These experiments support our contention that the T N F signaling pathway is a major mechanism of particle- induced NF-tiB activation. Nevertheless, the mechanism of rapid particle-induced T N F expression remains unclear. We hypothesize that the initial contact of particles with cell surface molecules activates intracellular signaling pathways that enhance T N F expression. In fact, Nakashima et al. [18] have proposed such a mechanism in which particle phagocytosis is not required for increased proinflammatory cytokine (TL-6, T N F ) expression and activation of the CD11 b/CD18 macrophage receptor mimics particlestimulated cytokine production. We have previously examined the mechanism of TNF-induced NF-tiB activation in osteoclast precursor cells [2]. T NF augments NF-tiB activity via a novel mechanism involving c-src tyrosine kinase-mediated phosphorylation of the inhibitor protein (ItiB) at residue 42. C-src, in this circumstance, forms a stable complex with ItiB, while NF-tiB is released and translocates to the nucleus. This activated NF-tiB dimer contains the p50 and p65 subunits, similar to that of particle-activated NF-tiB (Fig. 3 ) . Given that PMMA activates the transcription complex via TNF, a provocative hypothesis holds that this particle-induced event similarly involves tyrosine phosphorylation of ItiB. In fact, titanium-alloy particles not only stimulate T N F release

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from human macrophages but also activate NF-tiB by a mechanism requiring both tyrosine and serinehhreonine phosphorylation [18]. LPS, in the form of adherent endotoxin, is implicated in particle-induced osteolysis [5,23]. Others find that conimercially pure titanium particles and titanium implant surfaces have associated adherent endotoxin. In their system, titanium particle stimulation of macrophage cytokine production (IL-lfi, TNF), osteoclast differentiation and bone resorption is abolished after chemical removal of endotoxin. Thus, we tested the effects of PMMA particles on NF-tiB activity in LPS-hyporesponsive mice. In our system, the particleenhanced activity of NF-tiB was unchanged in LPShyporesponsive cells establishing that the event does not reflect endotoxin contamination. It remains to be determined, however, if particles and LPS activate NF-tiB by distinct signaling pathways. Both stimulate expression of T N F but are also capable of inducing other NF-tiB activating agents such as IL-1. Suggesting particles and LPS activate NF-tiB by different signaling pathways, LPS and titanium particles stimulate distinct protein tyrosine phosphorylation profiles [18] and IL-10 inhibits LPS-induced NF-tiB activation, while not impacting titanium particle activated NF-tiB [21]. PMMA activation of NF-KB,in osteoclast precursor cells, suggests this nuclear transcription factor participates in particle-stimulated osteoclastogenesis and osteolysis. Our data points toward T N F as an important cytokine mediator for PMMA particles, but other cytokines could be involved and different types of particles may excite distinct combinations of signaling pathways. Understanding of the specific molecular events that mediate NF-tiB activation by various particulates may identify specific common targets for therapeutic intervention.

Acknowledgements This paper was awarded the Orthopaedic Research Society Harris Award (2001). The study was supported by an OREF Research Grant (J.C.C.). National Institute of Health grants AR47096 (J.C.C.), AR32788 (S.L.T.), AR46523 (SLT), DE05413 (SLT), DE 13754 (Y.A.A.) and Arthritis Foundation Award (Y.A.A.). The authors thank Aline Stawizynski for assistance in manuscript preparation.

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