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Signaling Pathways for Tumor Necrosis Factor- and Interleukin-6 Expression in Human Macrophages Exposed to Titanium-Alloy Particulate Debris in Vitro*

YASUHARU NAKASHIMA, M.D., DOO-HOON SUN, M.D., MICHAEL C. D. TRINDADE, B.A., WILLIAM J. MALONEY, M.D., STUART B. GOODMAN, M.D., PH.D., DAVID J. SCHURMAN, M.D. and R. LANE SMITH, PH.D., STANFORD, CALIFORNIA

Investigation performed at the Orthopaedic Research Laboratory, Stanford University Medical Center, Stanford

	Abstract

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Background: Loosening of the implant after total joint arthroplasty remains a serious problem. The activation of macrophages by wear debris from implants, mediated by the release of cytokines that elicit bone resorption, may lead to loosening. The purpose of the present study was to elucidate the mechanisms of macrophage activation by titanium particles from the components of implants and to identify the signaling pathways involved in particle-mediated release of cytokines. Methods: Macrophages were isolated from mononuclear leukocytes obtained from healthy human donors and were exposed to titanium-alloy particles that had been obtained from periprosthetic membranes collected at revision total joint arthroplasties and then enzymatically prepared. The experimental protocols included examination of the effects of the inhibition of phagocytosis and the binding of antibodies to macrophage complement receptors on particle-induced macrophage activation. The release of the proinflammatory cytokines TNF- (tumor necrosis factor-alpha) and IL-6 (interleukin-6) was used to assess macrophage activation. The signaling pathways involved in the induction of cytokine release were analyzed by identification of phosphorylated proteins with use of the Western blot technique and by translocation of the transcription factors nuclear factor-kappa B (NF-B) and nuclear factor-interleukin-6 (NF-IL-6) into the nuclear protein fraction with use of electrophoretic mobility shift assays. The role of serine/threonine and tyrosine kinase pathways in the activation of nuclear factors and the release of cytokines was examined with use of selective pharmacological agents. Results: Exposure of macrophages to titanium-alloy particles in vitro for forty-eight hours resulted in a fortyfold increase in the release of TNF- and a sevenfold increase in the release of IL-6 (p < 0.01). Phagocytosis of particles occurred in approximately 73 percent of the macrophages within one hour of exposure. Pretreatment of the macrophages with cytochalasin B reduced phagocytosis by 95 percent but did not reduce the release of TNF- or IL-6. Thus, phagocytosis of particles was not necessary for induction of the release of TNF- or IL-6 in the cultured macrophages. Ligation of the macrophage CD11b/CD18 receptors by integrin-specific antibodies also increased the release of TNF- and IL-6. Antibodies to CD11b/CD18 receptors (macrophage Mac-1 receptors) reduced phagocytosis of particles by 50 percent (p < 0.05). (The CD11b/CD18 macrophage receptor is the macrophage receptor for the complement component CR3bi. The CD11b/CD18 macrophage receptor can also bind to ICAM-1 and ICAM-2. CD is the abbreviation for cluster of differentiation, and ICAM is the abbreviation for intercellular adhesion molecule.) Inhibition of phagocytosis was not accompanied by a decrease in the release of TNF- and IL-6. Blocking RNA synthesis with actinomycin D or preventing protein synthesis with cycloheximide abolished or decreased particle-induced release of TNF- and IL-6 from the macrophages. Macrophage release of TNF- and IL-6 in response to particles coincided with increased tyrosine phosphorylation and mitogen-activated protein kinase activation. Inhibition of tyrosine and serine/threonine kinase activity decreased the particle-induced release of cytokines. Exposure of macrophages to either titanium-alloy particles or to antibodies to the receptor proteins CD11b and CD18 for thirty minutes activated the transcription factors NF-B and NF-IL-6. Inhibition of particle phagocytosis did not block activation of the transcription factors. However, inhibition of tyrosine and serine/threonine kinase activity decreased the activation of NF-B and NF-IL-6. Conclusions: These data suggest that particle-induced macrophage release of TNF- and IL-6 does not require phagocytosis but is dependent on tyrosine and serine/threonine kinase activity culminating in activation of the transcription factors NF-B and NF-IL-6. Clinical Relevance: Retrieval studies have documented numerous macrophages in association with particulate debris in granulomatous tissue surrounding failed total joint replacements. However, the molecular basis on which wear particles induce macrophage expression of proinflammatory cytokines and bone-resorbing factors remains unclear. This in vitro study showed that particles incite the release of proinflammatory cytokines from macrophages in the absence of phagocytosis. These results imply that contact of wear particles with macrophage cell-surface membrane proteins, such as the complement receptor CD11b/CD18, is sufficient signal for release of proinflammatory cytokines. The data further suggest that release of proinflammatory cytokines follows transmission of a membrane recognition event through intracellular signaling pathways that effect gene activation and protein synthesis. Therefore, these data indicate that a reduction in the formation of wear particles can be expected to improve the outcome after total joint arthroplasty by decreasing macrophage activation.

	Introduction

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The longevity of a total joint replacement is limited by loosening, which may result from the biological reaction to particulate wear debris. The accumulation of wear particles at the site of an implant induces a granulomatous reaction and the release of cytokines that contribute to periprosthetic bone resorption^15,20,22,28. Histological examination of tissues from the sites of failed prostheses has revealed numerous macrophages, foreign-body giant cells, and abundant particulate wear debris^24,30,41,49. The granulomatous tissue results in periprosthetic bone resorption, loosening of the implant, and poor bone stock that decreases the potential for a successful revision operation.

Particle-induced inflammation and macrophage activation are associated with the loss of bone as a result of the formation of granulomatous tissue. Macrophage phagocytosis of polyethylene, polymethylmethacrylate, and titanium particles has been demonstrated in many studies of human tissues, animal models, and in vitro experiments^13,14,16,44. Maloney and Smith³⁰ showed that the macrophage response to particles results in the release of cytokines, such as TNF- (tumor necrosis factor-alpha), IL-6 (interleukin-6), IL-1 (interleukin-1 alpha), IL-1ß (interleukin-1 beta), and prostaglandin E₂. The levels of other inflammatory agents, such as the chemokines, matrix metalloproteinases, and lysosomal enzymes, also are elevated as part of the macrophage response to particulate debris^11,24,49. The localized release of these inflammatory molecules by macrophages or osteoclasts is believed to elicit bone resorption^39,47. Recent studies have shown that cyclic nucleotide signaling pathways are activated by polymethylmethacrylate and titanium particles and that pharmacological agents may modulate the release of inflammatory mediators^2,38.

The precise mechanisms by which particles of wear debris activate macrophages remain unclear. One hypothesis is that the particles interact with cell-surface receptors to trigger cellular activation. Macrophages possess multiple transmembrane receptors, including the complement receptors, Fc receptors, and nonspecific scavenger receptor molecules^6,17-19,40. Transmembrane receptors mediate macrophage function because the binding of receptor-specific antibodies inhibits phagocytosis^6,19,40. In addition, the binding of the antibodies to receptors increases the release of cytokines^{7,8,17,23,26,27,46}. These results support the concept that receptor-mediated events occur through particle-induced signaling pathways that activate gene expression.

The hypothesis for this particular study was that titanium-alloy particles interact with macrophage cell-surface receptors and, in the absence of phagocytosis, initiate the expression of cytokine genes (Fig. 1). In the present study, we determined the roles of phagocytosis, membrane-receptor binding, protein phosphorylation, and activation of transcription factors on macrophage expression of cytokines after exposure to titanium-alloy particles in vitro. The levels of TNF- and IL-6 were determined as an index of macrophage activation. The effects of the signaling pathways on the expression of TNF- and IL-6 were determined by an analysis of the effects of inhibitors of phagocytosis, protein kinases, transcriptional events, and protein synthesis.

View larger version (87K): [in this window] [in a new window]   Fig. 1 Schematic representation showing proposed signaling pathways of phagocytosis-independent macrophage activation by titanium particles. The hypothesis regarding particle-mediated activation of macrophages in the absence of phagocytosis supposes that membrane receptor proteins provide recognition events that are transmitted to the nucleus by way of intracellular signaling pathways. The intracellular signaling pathways involve phosphorylation of select target proteins that ultimately result in translocation of specific transcription factors from the cytoplasm to the nucleus. The transcription factors then modulate selective gene expression and result in the release of proinflammatory cytokines. PA = wear debris particle, ER = endoplasmic reticulum, TNF = tumor necrosis factor-, and CD11b/CD18 = epitope on macrophage receptor to which monoclonal antibodies bind.

	Materials and Methods

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Reagents
Monoclonal antibodies specific for CD11b, immunoglobulin 1A, and Fc receptor-1 were purchased from Biosource International (Camarillo, California). A monoclonal antibody specific for CD18 was purchased from Immunotech (Westbrook, Maine). Monoclonal antibodies specific for human phosphorylated tyrosine and mouse mitogen-activated protein kinase were purchased from Upstate Biotechnology (Lake Placid, New York). The antibody specific for mouse mitogen-activated protein kinase cross reacts with human mitogen-activated protein kinase. Human immunoglobulin-2 was purchased from Sigma Biosciences (St. Louis, Missouri). Ficoll-Paque was purchased from Pharmacia (Piscataway, New Jersey). Herbimycin, genistein, and staurosporin were purchased from Calbiochem-Novabiochem (La Jolla, California). H-7 was purchased from Seikagaku America (Rockville, Maryland). All other chemicals were obtained from Sigma Biosciences.

Preparation and Characterization of Particles
Titanium-alloy particles were obtained from granulomatous tissue surrounding failed titanium-alloy prostheses that had been removed at the time of revision operations²⁹. Briefly, formalin-fixed membranes were washed extensively with phosphate-buffered saline solution to remove residual formaldehyde, and subsequently they were divided into five-by-five-millimeter pieces. A papain suspension (seven micrograms per milliliter) in 0.05-molar sodium phosphate (pH 6.5) containing 0.02-molar N-acetyl cysteine was added, and the tissue samples were digested at 65 degrees Celsius overnight. After digestion, fragments of tissue were removed by filtration and the titanium particles were collected by centrifugation at 700 times gravity for twenty minutes. The particles were washed five times by repeated suspension and centrifugation in Dulbecco's phosphate-buffered saline solution. Energy-dispersive x-ray analysis showed that the elemental composition of the particles was consistent with titanium alloy²⁹. The mean size of the titanium particles was 0.7 micrometer²⁹. Isolated titanium-alloy particles were suspended in Dulbecco's phosphate-buffered saline solution and autoclaved. For testing, ten milliliters of this suspension of particles, which contained 400 billion particles per milliliter, was added to the cells on the basis of volumetric measure (percent volume per volume). All particle preparations were shown to be free of endotoxin by Limulus amoebocyte assay (Biowhittaker, Gaithersburg, Maryland).

Preparation of Cells
Adherent human peripheral blood monocytes were isolated from healthy individuals with use of Ficoll density-gradient fractionation. Briefly, cells from the buffy-coat layer of peripheral blood were dispersed in three volumes of phosphate-buffered saline solution. The suspended cells were layered over a solution of Ficoll-Paque (specific gravity, 1.077 grams per milliliter) and were centrifuged at 400 times gravity for thirty minutes at room temperature. The cellular interface that contained the mononuclear cells was collected by aspiration, and the cells were washed three times with fifty milliliters of cold (4-degree-Celsius) Dulbecco's phosphate-buffered saline solution. The monocytes were resuspended in RPMI-1640 medium containing 5 percent human serum and twenty-five micrograms of gentamicin per milliliter and were plated on 100-millimeter tissue-culture dishes in eight milliliters of medium at a cell density of 5 x 10⁵ cells per centimeter². The cells were cultured for twenty-four hours to allow cell adhesion. The medium then was removed, and the cultures were washed with Dulbecco's phosphate-buffered saline solution (three times, with use of ten milliliters for each wash) to remove nonadherent cells. More than 90 percent of the adherent monocytes behaved as macrophages and exhibited nonspecific esterase staining.

Treatment of Macrophages with Particles
The medium was removed, and the adherent macrophages were exposed to a 0.075 percent volume per volume suspension of titanium particles in ten milliliters of medium containing 400 billion particles per milliliter. The particles remained with the cells throughout the exposure periods. This particle concentration was previously determined to induce substantial levels of cytokine production. An increase in the particle concentration did not further increase production of these cytokines in macrophages. Tests for release of TNF- and IL-6 from macrophages were carried out in RPMI-1640 medium containing 5 percent human serum in the presence of antibiotics. For experiments with selective inhibitors, macrophages were pretreated with specific pharmacological agents and the pretreatment period was followed by exposure to titanium particles. The concentrations of the inhibitors were determined, with use of a test of cell viability, to be nontoxic and were selected to maintain specificity of action with respect to human macrophages. The addition of the bacterial endotoxin lipopolysaccharide at a concentration of 100 nanograms per milliliter served as a positive control for macrophage activation.

Phagocytosis Assay
Phagocytosis of the particles was analyzed with use of a modification of the methods described previously⁶. Titanium-alloy particles were added to macrophage cultures, and the cultures were incubated for one hour with or without exposure to antibodies to cell-surface receptors or pretreatment with cytochalasin B. After incubation, the cells were washed with Dulbecco's phosphate-buffered saline solution (five times, with use of ten milliliters for each wash) to remove nonphagocytosed particles. The cells were trypsinized, removed from the plates, and counted with use of a hematocytometer. The number of cells phagocytosing particles was quantified by counting 200 cells per well, and the data were expressed as a percentage of cells containing particles.

Analysis of the Release of TNF- and IL-6
Macrophages were exposed to titanium-alloy particles and cell-surface receptor antibodies with or without protein kinase inhibitors. Samples from the culture medium were collected at various time-periods and stored at -20 degrees Celsius until use. The levels of TNF- and IL-6 were measured with use of commercially available enzyme-linked immunosorbent assay kits (R and D Systems, Minneapolis, Minnesota).

Northern Blot Analysis
Total RNA was extracted from cells with use of a solution containing four-molar guanidine isothiocyanate, twenty-five-millimolar sodium citrate, 0.2 percent (weight per volume) n-lauroylsarcosine sodium, and 0.7 percent (volume per volume) ß-mercaptoethanol³. Total RNA (ten micrograms) from each sample was separated on a formaldehyde agarose gel and transferred by capillary blotting overnight to a nylon membrane. Complementary DNA fragments for TNF- and IL-6 were labeled with ³²phosphate by random priming (Amersham International, Bucks, United Kingdom). Each probe had a specific activity between 4 to 6 x 10⁸ counts per minute per microgram. Blots were hybridized at 42 degrees Celsius overnight and washed at 55 degrees Celsius in a 0.5-times-standard concentration of saline-sodium citrate solution with 0.1 percent sodium dodecyl sulfate for one hour. The membranes were exposed to Kodak X-Omat film (Eastman Kodak, Rochester, New York) at -80 degrees Celsius in the presence of a light-intensifying screen (DuPont, Wilmington, Delaware).

Detection of Tyrosine Phosphorylation and Mitogen-Activated Protein Kinase by Western Immunoblot Analysis
A modification of the methods described by Weinstein et al. was used⁴⁸. Macrophages (10⁷ cells per thirty-five-millimeter dish) were challenged with particles or antibodies. After treatment, the cells were washed with Dulbecco's phosphate-buffered saline solution (three times,with use of ten milliliters for each wash) containing one-millimolar sodium orthovanadate and were scraped into 300 microliters of lysis buffer (1 percent t-octylphenoxypolyethoxyethanol, twenty-millimolar Tris[hydroxymethyl]aminomethane hydrochloride [pH 8.0], 137-millimolar sodium chloride, 10 percent glycerol, one-millimolar sodium orthovanadate, two-millimolar EDTA, one-millimolar phenylmethanesulfonyl fluoride, twenty-micromolar leupeptin, and 0.15 unit of aprotinin per milliliter). The lysate was placed on ice for twenty minutes and then was centrifuged at 12,000 revolutions per minute for fifteen minutes at 4 degrees Celsius. The solubilized protein was collected and measured with use of the Bradford procedure (Bio-Rad Laboratories, Emeryville, California). The protein concentration then was adjusted to two milligrams per milliliter in 62.5-millimolar Tris[hydroxymethyl]aminomethane hydrochloride (pH 6.8), 2.3 percent sodium dodecyl sulfate, 100-millimolar dithiothreitol, and 0.005 percent bromphenol blue and boiled. The proteins (twenty micrograms) were electrophoresed on a 10 percent polyacrylamide gel and were transferred to Immobilon-P membrane (Millipore, Bedford, Massachusetts) in electroblotting buffer (twenty-millimolar Tris[hydroxymethyl]aminomethane hydrochloride [pH 8.0], 150-millimolar glycine, and 20 percent methanol) for two hours at twenty-four volts. The membranes were blocked in a milk solution (2 percent nonfat dry milk, 150-millimolar sodium chloride, fifty-millimolar Tris[hydroxymethyl]aminomethane hydrochloride, 0.05 percent polyoxyethylenesorbitan monolaurate, and 0.02 percent sodium azide) at room temperature for two hours. The blots were exposed to a monoclonal antibody against phosphorylated tyrosine or a polyclonal antibody against mitogen-activated protein kinase and subsequently were developed with use of goat anti-immunoglobulin alkaline phosphatase in conjunction with an enhanced chemiluminescence Western blot detection system (Bio-Rad Laborato ries).

Preparation of Nuclear Extracts and Electrophoretic Mobility Shift Assay
Nuclear extracts were prepared as previously described⁹. All buffers were supplemented with phenylmethanesulfonyl fluoride (0.5 millimolar) and aprotinin, pepstatin A, and leupeptin (five micrograms per milliliter each). Briefly, cells were washed three times with washing buffer (ten-millimolar Tris[hydroxymethyl]aminomethane hydrochloride [pH 7.5], 0.13-molar sodium chloride, five-millimolar potassium chloride, and eight-millimolar magnesium chloride) and then scraped into hypotonic buffer (twenty-millimolar N-2-hydroxyethylpiperazine-N'-2-ethane sulfonic acid-potassium hydroxide [pH 7.9], five-millimolar potassium chloride, and 0.5-millimolar magnesium chloride). The suspensions were homogenized with Dounce homogenizer (B pestle), and the crude nuclei were collected by centrifugation at 600 times gravity for ten minutes. The nuclei were suspended in extraction buffer (twenty-millimolar N-2-hydroxyethylpiperazine-N'-2-ethane sulfonic acid-potassium hydroxide [pH 7.9], 25 percent glycerol, 0.5-molar sodium chloride, 1.5-millimolar magnesium chloride, and 0.2-millimolar EDTA-sodium hydroxide [pH 8.0]), sonicated, and centrifuged at 20,000 times gravity for thirty minutes at 4 degrees Celsius. Titanium-alloy particles were removed as a pellet. The nuclear proteins were collected in the supernatant fraction, and the protein concentrations were determined with the Bio-Rad protein assay. A typical yield was 100 to 200 micrograms of protein. Synthetic double-stranded oligonucleotides of the following sequences were end-labeled with ³²P: 5'-AGT TGA GGG GAC TTT CCC AGG C-3' and 3'-TCA ACT CCC CTG AAA GGG TCC G-5' for NF-B (nuclear factor-kappa B) and 5'-AGCT TTA AGA TTG CAC AAT GTG ACG TCA-3' and 3'-AAT TCT AAC GTG TTA CAC TGC AGT TCG A-5' for NF-IL-6 (nuclear factor-interleukin-6).

The DNA-protein binding reaction was performed for twenty minutes in a volume of ten microliters. The reaction mixture contained ten micrograms of nuclear protein, fifty micrograms of polydeoxyinosinic-deoxycytidylic acid per milliliter, ten-millimolar Tris[hydroxymethyl]aminomethane hydrochloride (pH 7.5), 100-millimolar sodium chloride, one-millimolar EDTA, one-millimolar dithiothreitol, 20 percent glycerol, and 50,000 disintegrations per minute ³²P end-labeled oligonucleotide. After incubation, the samples were loaded onto a 4 percent polyacrylamide gel running at 100 volts. After three hours, the gels were subjected to autoradiography for analysis.

Statistical Analysis
The statistical analysis was performed with use of analysis of variance and the Student two-sample (two-tailed) t test. P values of less than 0.05 were considered significant.

	Results

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Macrophage Phagocytosis of Titanium-Alloy Particles
Experiments first were carried out to determine the effects of inhibiting phagocytosis with cytochalasin B or binding (ligating) antibodies to CD11b and CD18 receptors (or the CD11b/CD18 macrophage receptor) on the uptake of titanium particles by macrophages. In the absence of any agent, a mean (and standard error of the mean) of 72.5 ± 6.0 percent of macrophages contained particles after a one-hour period of exposure (Table I). Pretreatment of the cells with cytochalasin B essentially abolished phagocytosis and reduced the percentage of cells containing particles to 5.8 ± 0.9 (p < 0.002). Ligation of either CD11b or CD18 with a specific antibody decreased the percentage of macrophages containing particles to 51.0 ± 3.7 (p < 0.05) and 48.5 ± 2.7 (p < 0.05), respectively. Ligation with a combination of antibodies to CD11b and CD18 decreased the percentage of macrophages containing particles to 36.5 ± 4.9 (p < 0.05).

Induction of Cytokine Expression by Titanium-Alloy Particles
Experiments were carried out to demonstrate the effect of titanium particles on the release of TNF- and IL-6. In the absence of titanium particles, the levels of TNF- and IL-6 reached only fifty and thirty-five picograms per milliliter, respectively, after forty-eight hours of culture. In the presence of titanium particles, the macrophages exhibited a time-dependent increase in the release of TNF- and IL-6 that was first observed at six hours and plateaued at twenty-four hours (Figs. 2, A, and 3, A). In the presence of particles, the release of TNF- increased from 500 picograms per milliliter at six hours to 2000 picograms per milliliter at twenty-four hours and that of IL-6 increased from 115 picograms per milliliter at twelve hours to 250 picograms per milliliter at twenty-four hours (p < 0.01). When phagocytosis of titanium particles was inhibited with cytochalasin B, induction of TNF- and IL-6 synthesis and secretion remained unchanged from that found when the macrophages were exposed to titanium particles alone (Figs. 2, B, and 3, B). Inhibition of protein and RNA synthesis with cycloheximide and actinomycin D, respectively, reduced or completely abolished the release of both cytokines in response to titanium particles (Figs. 2, B, and 3, B).

View larger version (28K): [in this window] [in a new window]   Fig. 2 Graphs showing the effects of titanium particles on the release of TNF- into culture medium. *p < 0.05 compared with control, as determined with analysis of variance and the Student two-sample (two-tailed) t test. A: Macrophages exposed to titanium particles for various time-periods. B: Macrophages exposed to titanium particles for twenty-four hours. These included macrophages exposed to titanium particles (Ti) only, macrophages pretreated with cytochalasin B (CytoB) at ten micromolar for ten minutes, macrophages pretreated with cycloheximide (CHX) at twenty micrograms per milliliter for three hours, and macrophages pretreated with actinomycin D (Act-D) at ten micrograms per milliliter for three hours. CTL = control (macrophages incubated without particles).

View larger version (26K): [in this window] [in a new window]   Fig. 3 Graphs showing the effect of titanium particles on the release of TNF- into culture medium. *p < 0.05 compared with control, as determined with analysis of variance and the Student two-sample (two-tailed) t test. A: Macrophages exposed to titanium particles for various time-periods. B: Macrophages exposed to titanium particles for twenty-four hours. These included macrophages exposed to titanium particles (Ti) only, macrophages pretreated with cytochalasin B (CytoB) at ten micromolar for ten minutes, macrophages pretreated with cycloheximide (CHX) at twenty micrograms per milliliter for three hours, and macrophages pretreated with actinomycin D (Act-D) at ten micrograms per milliliter for three hours. CTL = control (macrophages incubated without particles).

Effects of Titanium-Alloy Particles on Cytokine Messenger RNA Signal Levels
Experiments then were performed to determine if transcriptional events were involved in the macrophage response to titanium particles. The TNF- and IL-6 messenger RNA signal levels were undetectable with Northern blot analysis of macrophages cultured in the absence of titanium particles (Fig. 4). However, TNF- and IL-6 messenger RNA signal levels were evident after the macrophages had been exposed to titanium particles for four hours. Treatment with cytochalasin B had no effect on induction of messenger RNA signals for TNF- and IL-6 by titanium particles. These observations suggest that induction of TNF- and IL-6 synthesis and release involves mechanisms that do not require phagocytosis.

View larger version (81K): [in this window] [in a new window]   Fig. 4 Northern blot analysis showing messenger RNA levels of TNF- and IL-6. Total RNA was isolated after four hours and hybridized to TNF- and IL-6 cDNA probes. RNA load was determined by ethidium bromide labeling of 18S RNA. CTL = control (macrophages incubated without particles), Ti = macrophages exposed to titanium particles (0.075 percent volume per volume), and Ti + CytoB = macrophages pretreated with cytochalasin B at ten micromolar for ten minutes followed by exposure to titanium particles at 0.075 percent volume per volume.

Effects of Membrane Receptor Antibodies on Cytokine Expression
Activation of macrophages in the absence of particle phagocytosis could occur by processes involving membrane receptor-mediated signaling. Therefore, experiments were carried out to determine if the binding of a protein (the ligand) to its receptor would suffice as a stimulus for macrophage release of cytokines. Antibodies to the receptor proteins CD11b and CD18 were chosen as the specific ligands, and the complement receptor (CD11b/CD18) was selected for macrophage activation. (The CD11b/CD18 macrophage receptor is the macrophage receptor for the complement component CR3bi. The CD11b/CD18 macrophage receptor can also bind to ICAM-1 and ICAM-2. CD is the abbreviation for cluster of differentiation, and ICAM is the abbreviation for intercellular adhesion molecule.) The addition of antibodies to the complement receptor proteins CD11b and CD18 increased release of TNF- and IL-6 (Fig. 5, A and B). In contrast, the addition of antibodies to immunoglobulin or to the Fc receptor had no effect on the release of cytokines. Cytochalasin B had no effect on induction of macrophage release of cytokines by antibody to CD11b. Experiments then were carried out to define the mechanisms of signal transduction that elicit synthesis and release of TNF- and IL-6 in response to titanium particles.

View larger version (26K): [in this window] [in a new window]   Fig. 5 Graphs showing the effect of the addition of cell-surface receptor antibodies on the release of TNF- (A) and IL-6 (B) from macrophages. Macrophages were treated with cell-surface receptor antibodies or immunoglobulin for two hours. CTL = control (macrophages incubated without particles), CD11b Ab = CD11b antibody (five micrograms per milliliter), CD18 Ab = CD18 antibody (five micrograms per milliliter), Fc Ab = Fc-receptor-1 antibody (five micrograms per milliliter), IgG = immunoglobulin 1A (five micrograms per milliliter), and CD11b Ab + CytoB = CD11b Ab with pretreatment with cytochalasin B.

Protein Kinase Inhibitors and Cytokine Release in Response to Titanium-Alloy Particles
The tyrosine kinase inhibitors, herbimycin and genistein, showed a dose-dependent inhibition of both TNF- and IL-6 release (Fig. 6, A and B). The serine/threonine kinase inhibitor, H-7, also decreased release of TNF- and IL-6 in a dose-dependent manner. In contrast, at nontoxic concentrations, the protein kinase-C inhibitor, staurosporin, did not alter particle-induced production of TNF- and IL-6.

View larger version (39K): [in this window] [in a new window]   Fig. 6 Graphs showing the effect of protein kinase inhibitors on the release of TNF- (A) and IL-6 (B). Macrophages were pretreated with herbimycin A for four hours and with genistein, H-7, or staurosporin (Stauro) for two hours at the concentrations indicated. The cells then were exposed to titanium particles at 0.075 percent volume per volume for twenty-four hours in the presence of the inhibitors. CTL = control (macrophages incubated without particles), and Ti = titanium. *p < 0.05 compared with control, as determined with analysis of variance and the Student two-sample (two-tailed) t test.

Tyrosine Phosphorylation
Western blot analysis demonstrated that titanium-alloy particles increased tyrosine phosphorylation of a number of distinct macrophage proteins. Differences in tyrosine phosphorylation patterns were evident between control and particle-challenged cells for proteins migrating with electrophoretic mobilities of seventy-eight, fifty-three, forty-five, and twenty kilodaltons. Phosphorylation of proteins at forty-five and twenty kilodaltons was apparent within thirty minutes of exposure to particles, but the signal was decreased by four hours after exposure to particles (Fig. 7, A). The profiles for particle-induced tyrosine phosphorylation were different from those induced after exposure of the cells to lipopolysaccharide (Fig. 7, B).

View larger version (52K): [in this window] [in a new window]   Fig. 7 Western blot analysis showing kinetics of protein-tyrosine phosphorylation induced by titanium particles (A) and by lipopolysaccharide (B). Macrophages were unchallenged (A and B, lane 1), challenged with titanium particles at 0.075 percent volume per volume (A, lanes 2 through 6), or exposed to lipopolysaccharide at 100 nanograms per milliliter (B, lanes 2 through 6). Cells from both treatment groups were lysed at thirty minutes (lane 2), sixty minutes (lane 3), 120 minutes (lane 4), 240 minutes (lane 5), and 480 minutes (lane 6). Differences in tyrosine phosphorylation patterns were evident between control and particle-challenged cells for proteins migrating with electrophoretic mobilities of seventy-eight, fifty-three, forty-five, and twenty kilodaltons (A, arrows). Differences were evident in posphorylated proteins after exposure to lipopolysaccharide (B, arrows).

Expression of Mitogen-Activated Protein Kinase
Localization of tyrosine phosphorylated proteins to the forty-five-kilodalton region of the gel suggested that mitogen-activated protein kinase was involved in the macrophage response to titanium-alloy particles. Western blot analysis revealed that levels of mitogen-activated protein kinase increased in a time-dependent manner during the first sixty minutes after the addition of particles (Fig. 8). Trace levels of mitogen-activated protein kinase were observed in the cells in the absence of particles. The pattern of increased levels of mitogen-activated protein kinase was similar to the cellular response after the addition of antibody to the CD11b receptor (Fig. 8).

View larger version (34K): [in this window] [in a new window]   Fig. 8 Western blot analysis showing the effects of titanium particles and CD11b antibody on expression of mitogen-activated protein kinase. Macrophages were challenged with titanium particles (Ti) at 0.075 percent volume per volume and CD11b antibody (ten micrograms per milliliter), and whole cell lysates were prepared at the time-periods indicated. CTL = control.

Translocation of Transcription Factors
Inhibition of particle-induced release of cytokines by actinomycin D suggested that transcriptional activation was involved. Tyrosine phosphorylation and the mitogen-activated protein kinase pathway are linked to activation of nuclear proteins that translocate to the nucleus and induce gene expression. In the present study, two transcription factors, NF-B and NF-IL-6, which are closely associated with increased expression of the TNF- and IL-6 genes, were analyzed with use of electrophoretic mobility shift assays. Levels of NF-B increased within thirty minutes of exposure to particles and remained relatively constant over a period from sixty minutes to six hours (Fig. 9, A). The increased level of NF-B in the nucleus in response to particles was not influenced by pretreatment of the cells with cytochalasin B (Fig. 9, B). Particle activation of NF-B was as effective as the addition of antibodies to CD11b and CD18 (Fig. 9, C). Antibody to immunoglobulin and the Fc receptor did not induce translocation of NF-B.

View larger version (45K): [in this window] [in a new window]   Fig. 9 Western blot analysis showing the time-course for the activation of NF-B by titanium particles. A: Nuclear proteins from unchallenged macrophages (lane 1) and macrophages challenged with titanium-alloy particles at 0.075 percent volume per volume for thirty minutes (lane 2), sixty minutes (lane 3), 120 minutes (lane 4), and 240 minutes (lane 5), and 360 minutes (lane 6). An extract from cells challenged with particles for sixty minutes was reacted with specific competitor oligonucleotide (lane 7) and nonspecific competitor oligonucleotide (lane 8). B: Cultures pretreated with cytochalasin B at ten micromolar for ten minutes and then challenged with titanium particles for two hours. The cells were unchallenged (lane 1), challenged with titanium particles (lane 2), or challenged with titanium particles after pretreatment with cytochalasin B (lane 3). C: Macrophages challenged with antibodies or immunoglobulin for two hours. The cells were unchallenged (lane 1) or they were challenged with titanium particles (lane 2), CD11b antibody (five micrograms per milliliter) (lane 3), CD18 antibody (five micrograms per milliliter) (lane 4), Fc-receptor-1 antibody (five micrograms per milliliter) (lane 5), immunoglobulin-1A (five micrograms per milliliter) (lane 6), or CD11b after pretreatment with cytochalasin B (lane 7).

View larger version (53K): [in this window] [in a new window]   Fig. 10 Western blot analysis showing the time-course for the activation of NF-IL-6 by titanium particles. A: Nuclear proteins from unchallenged macrophages (lane 1) and macrophages challenged with titanium-alloy particles at 0.075 percent volume per volume for thirty minutes (lane 2), sixty minutes (lane 3), 120 minutes (lane 4), 240 minutes (lane 5). The nuclear extract from macrophages challenged with particles for sixty minutes was reacted with specific competitor oligonucleotide (lane 6) and nonspecific competitor oligonucleotide (lane 7). B: Cultures pretreated with cytochalasin B at ten micromolar for ten minutes and then challenged with titanium particles for two hours. The cells were unchallenged (lane 1), challenged with titanium particles (lane 2), or challenged with titanium particles after pretreatment with cytochalasin B (lane 3). C: Macrophages challenged with antibodies or immunoglobulin for two hours. The cells were unchallenged (lane 1) or they were challenged with titanium particles (lane 2), CD11b antibody (five micrograms per milliliter) (lane 3), CD18 antibody (five micrograms per milliliter) (lane 4), Fc-receptor-1 antibody (five micrograms per milliliter) (lane 5), immunoglobulin-1A (five micrograms per milliliter) (lane 6), or CD11b after pretreatment with cytochalasin B (lane 7).

View larger version (49K): [in this window] [in a new window]   Fig. 11 Western blot analysis showing the effects of protein kinase inhibitors on activation of NF-B (A) and NF-IL-6 (B) in response to titanium particles. Macrophages were pretreated with herbimycin A at one micromolar for four hours, genistein at 100 micromolar for two hours, H-7 at 100 micromolar for two hours, and staurosporin at 100 nanomolar for two hours. After a subsequent two-hour exposure to titanium particles (0.075 percent volume per volume), the nuclear proteins were extracted and analyzed. The cells were unchallenged (lane 1), challenged with titanium particles without inhibitors (lane 2), or challenged with titanium particles after pretreatment with herbimycin A (lane 3), after pretreatment with genistein (lane 4), after pretreatment with H-7 (lane 5), or after pretreatment with staurosporin (lane 6).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
In the present study, titanium particles induced the synthesis and secretion of TNF- and IL-6 by macrophages in vitro. The release of TNF- and IL-6 occurred in the presence of cytochalasin B, which inhibits phagocytosis; thus, it appears that phagocytosis was not required. Induction of TNF- and IL-6 release also occurred after the binding of antibodies to the macrophage complement-receptor proteins CD11b and CD18. The CD11b and CD18 antibodies mimic the effects of complement or complement fragments.

The similarity between activation of macrophages through the complement receptor and activation by titanium particles in the absence of phagocytosis suggests that particulate debris from orthopaedic biomaterials may bind plasma components, such as complement proteins or fragments of proteins. The release of TNF- and IL-6 in response to titanium particles required gene expression and was dependent on kinase activity and transcription factor activation. Particle-induced activation of transcription factors was similar to that observed after the binding of complement receptor proteins and was independent of phagocytosis.

Macrophages were challenged with titanium particles isolated from periprosthetic tissues of failed total hip replacements to provide particles of a size, morphology, and composition generated in vivo^29,44. The target cells chosen for particle stimulation were human monocytes, as these cells are precursors of tissue macrophages. In culture, macrophages exposed to particles of titanium and cobalt-chromium alloy exhibit phagocytosis of particles within the cells that coincides with an increased cytoplasmic volume³¹. In the present study, particle phagocytosis occurred within one hour after exposure. In alveolar macrophages, phagocytosis takes place within a similar time-period and is influenced more by opsonization of particles than by the type of particle^17,19,40. The effect of cytochalasin B on phagocytosis of titanium particles was similar to that observed for alveolar macrophages, primary human macrophages, and transformed macrophage cell lines^21,35,42.

The inhibition of phagocytosis of titanium particles by antibodies to the complement receptor protein CD11b or CD18 suggests that complement surface receptors are involved in the recognition of titanium particles. Inhibition of phagocytosis of the titanium particles was more pronounced with both antibodies, suggesting that membrane-receptor accessibility may be involved. The observation that macrophages were activated either by antibodies to complement receptor proteins or by particles in the presence of cytochalasin B shows that phagocytosis is not required for induction of the release of proinflammatory cytokines. As shown in the present study, the release of cytokines from activated macrophages was dependent on transcriptional and translational processes as confirmed by the inhibitory effects of actinomycin D and cycloheximide, respectively.

The fact that phagocytosis was not necessary suggests that particle activation of macrophages may be influenced by opsonization of plasma components, such as complement or complement fragments³². In the present study, opsonization of particles may have occurred in vivo before isolation or in culture in the presence of human serum. These data imply that binding of titanium particles to membrane surface ligands is sufficient for cellular activation. However, the size of the particles may influence membrane-receptor recognition so that activation cannot occur above or below a certain size-threshold. These phenomena may be related to protein conformation states that are subject to the physiochemical states of orthopaedic biomaterials or to the distribution and number of cellular membrane receptors.

As activation of gene transcription depends on receptor kinase activity, the effects of titanium-alloy particles on macrophage protein tyrosine phosphorylation were determined with the Western blot technique. Exposure to particles induced the appearance of newly phosphorylated proteins within thirty minutes, but they were no longer evident after four hours. Phosphorylated proteins were evident at seventy-eight and twenty kilodaltons in cells exposed to particles, but they were not detected in cells exposed to bacterial lipopolysaccharide⁵. The data suggest that different signaling pathways are involved in macrophage activation by either particles or bacterial antigens³⁶. Analysis of the effect of pharmacological agents on particle-induced release of cytokines at the level of cyclic nucleotide and cyclic nucleotide-protein kinase demonstrates differential effects of agonists and antagonists depending on the cytokine in question^2,38. For example, Ritchie et al.³⁸ found that dibutyl cAMP inhibits titanium particle-induced release of TNF- but increases titanium particle-induced release of IL-6. These data suggest that multiple signaling pathways are involved for different inflammatory mediators in a single type of cell.

Analysis of macrophage lysates with the Western blot technique showed that titanium particles activated mitogen-activated protein kinase within fifteen minutes of exposure, as evidenced by tyrosine phosphorylated proteins at the forty-five-kilodalton region^1,37. Mitogen-activated protein kinase is activated by a mitogen-activated protein kinase kinase that is a tyrosine and a serine/threonine kinase¹⁰. Specific antibody detection of mitogen-activated protein kinase showed that the protein was evident at thirty, forty-five, and sixty minutes, suggesting a decrease in the rapid turnover of this protein. The level of mitogen-activated protein kinase also was increased within fifteen minutes after exposure to the CD11b antibody, but the level decreased within sixty minutes. This finding may reflect either degradation of the antibody or increased turnover of the mitogen-activated protein kinase.

Because tyrosine phosphorylation and mitogen-activated protein kinase expression result in the modification of proteins that influence gene transcription, the levels of two transcription factors associated with expression of TNF- and IL-6, NF-B and NF-IL-6, were determined with use of electrophoretic mobility shift assays^{4,12,37,43,45}. The hypothesis was that increased expression of TNF- and IL-6 depends on particle-mediated translocation of these transcription factors into the nucleus. Activation of NF-B precedes expression of TNF-, IL-1, and IL-1ß in a variety of systems^4,12,43,45. Factors activating NF-B in macrophages include bacterial polysaccharides, inflammatory cytokines, and phorbol esters. Similarly, activation of NF-IL-6 coincides with increased release of IL-6 in macrophages, fibroblasts, osteoblasts, and neutrophils^25,33,34. As shown in the present study, titanium-alloy particles activated NF-B and NF-IL-6 within one hour after exposure. Activation of NF-B and NF-IL-6 was not influenced by the pretreatment of the cells with cytochalasin B. In contrast, inhibitors of tyrosine and serine/threonine kinases decreased the activation of NF-B and NF-IL-6. The decreased activation of the transcription factors coincided with decreased levels of TNF- and IL-6 in the culture medium in the presence of the kinase inhibitors. Multiple signaling pathways may be involved, as both types of inhibitors decreased particle-induced activation of transcription factors and the release of cytokines.

In summary, the present study showed that macrophage activation by titanium particles for the induction of increased expression of proinflammatory cytokines does not require phagocytosis. In other systems, receptor molecules undergo conformational changes that lead to activation of intracellular signaling proteins^23,26,27,46. As shown in the present study, titanium particles activate macrophage NF-B and NF-IL-6 and increase the release of TNF- and IL-6, an effect that is similar to that of antibody ligation of CD11b or CD18. The data suggest that cellular contact with wear particles from orthopaedic implants is sufficient to induce the release of cytokines that may determine the outcome of total joint arthroplasty.

	Footnotes

 
*Although none of the authors has received or will receive benefits for personal or professional use from a commercial party related directly or indirectly to the subject of this article, benefits have been or will be received, but are directly solely to a research fund, foundation, educational institution, or other nonprofit organization with which one or more of the authors is associated. Funds were received in total or partial support of the research or clinical study presented in this article. The funding sources were the Stanford Orthopaedic Research Fund, the Stanford Orthopaedic Nickel Fund, and Wright Medical.

Orthopaedic Research Laboratory, Room 144, Stanford University Medical Center, 300 Pasteur Drive, Stanford, California 94305-5341.

Department of Orthopaedics, Washington University School of Medicine, One Barnes Hospital Plaza, St. Louis, Missouri 63110.

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