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© 1996 The Journal of Bone and Joint Surgery, Inc.
Increased Levels of Tumor Necrosis Factor-
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and Interleukin-6 Protein and Messenger RNA in Human Peripheral Blood Monocytes due to Titanium Particles*
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Abstract |
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Cytokines produced by macrophages in the periprosthetic membranes surrounding joint replacements have been implicated as causal agents in osteolysis and prosthetic loosening. The present study characterizes the response of human peripheral blood monocytes to titanium particles. Monocytes were obtained from volunteers and blood that had been donated to the American Red Cross and were cultured in the presence of titanium particles (one to three micrometers in diameter). There were consistent dose-dependent increases in the production of TNF-
(tumor necrosis factor-) and IL-6 (interleukin-6) protein, with the greatest stimulation generally observed with a concentration of 6 x 105 to 6 x 106 particles of titanium per milliliter. The level of TNF- was the greatest (fifty to 1000 times greater than the control level) after eight hours of exposure to titanium particles; the level of IL-6 was two to five times greater than the control level after sixteen hours of exposure. These increases were similar to those observed after stimulation with lipopolysaccharide and depended on de novo synthesis rather than on release from intracellular stores. The production of TNF- was inhibited in a dose-dependent manner by the translational inhibitor cycloheximide and the transcriptional inhibitor actinomycin D, indicating the requirement for both mRNA (messenger RNA) and protein synthesis for the induction of cytokine synthesis by titanium particles. Although the increase in the levels of cytokine mRNA in response to titanium was rapid (thirty to ninety minutes), the increase in the level of TNF- mRNA preceded that of IL-6 mRNA. The level of TNF- mRNA was the greatest at ninety minutes and the level of IL-6 mRNA was the greatest at three hours. After stimulation with titanium particles, the level of TNF- mRNA was increased as much as fivefold and the level of IL-6 mRNA, as much as twelvefold.
CLINICAL RELEVANCE: Awareness of the importance of wear debris particles in cytokine-induced bone resorption has resulted in improvements in the designs of implants and in operative techniques to reduce wear of components. The present study further elucidates the biological mechanisms involved in periprosthetic osteolysis. Titanium-stimulated biosynthesis of the cytokines TNF- and IL-6, which are both potent stimulators of bone resorption, requires increases in the synthesis of both mRNA and protein by monocytes. An understanding of the complex mechanisms of the induction of cytokine synthesis by particles of wear debris will facilitate the design of pharmacological agents to control periprosthetic bone resorption. These agents, in combination with other efforts to reduce the generation of wear debris, may improve the longevity of orthopaedic implants. |
Introduction |
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Periprosthetic osteolysis and loosening after joint arthroplasty continue to be serious problems in the operative treatment of arthritis and osteoarthrosis. A recent study of 121 total hip arthroplasties without cement, followed for a mean of sixty-seven months (range, fifty-five to seventy-nine months), revealed radiographic evidence of loosening of the femoral component in eleven hips and evidence of osteolysis around nine (8 per cent) of 110 stably fixed femoral components24. Examination of the tissue from the bone-implant interface from patients who have aseptic loosening has shown characteristic histological features consisting of numerous macrophages and giant cells laden with particles of debris6,10,22. Moreover, particles of titanium debris were found in 63 and 75 per cent of membranes retrieved from patients managed with revision arthroplasty in two recent studies7,23. These observations suggest that titanium wear debris may play a substantial role in aseptic loosening.
Although the mechanism by which particles of debris lead to osteolysis is unknown, the secretion of cytokines by macrophages has been implicated in this process. Immunohistochemical analysis of periprosthetic membranes and explant cultures of specimens of recovered membranes have demonstrated the presence of cytokines2,8,19, and in situ hybridization studies have localized cytokine mRNA (messenger RNA) to macrophages present within the membrane18. Furthermore, cytokines secreted by macrophages have been shown to be associated with bone resorption in culture1,3,4,12.
Particles of debris stimulate the production of cytokines in cell cultures of murine and rodent monocytes and macrophages. In one study of rodent peritoneal macrophages exposed to particles of debris from Ti-Al-V alloy, the activity of TNF- (tumor necrosis factor-), IL-1 (interleukin-1), and IL-6 (interleukin-6) increased threefold to tenfold15. In murine peritoneal macrophages exposed to titanium particles, a fivefold increase in IL-1 and a tenfold increase in prostaglandin E2 were observed12. When murine macrophages were exposed to cement (polymethylmethacrylate) debris, they synthesized five times more TNF- than was seen in control cultures16.
Many of the previous investigations of the effects of debris particles on the synthesis of cytokines have been performed in cell lines or in animal macrophage models, with the assumption that human cells behave in a similar fashion. This hypothesis has only recently been tested with human peripheral blood monocytes14,17, the precursors of tissue macrophages, with which they share immunological, phenotypical, and biochemical characteristics25. In areas of inflammation, peripheral blood monocytes enter tissue, where they initiate phagocytosis and macrophage function25. Thus, human peripheral blood monocytes are perhaps the most valid model for investigation of the effects of debris particles on the production of cytokines.
The two goals of the present study were to examine the effect of debris particles on the production of TNF- and IL-6 in human peripheral blood monocytes and to determine the mechanism by which the production of these cytokines is induced. The study demonstrated a rapid induction of TNF- and IL-6 production in peripheral blood monocytes, an effect dependent on the de novo synthesis of both mRNA and protein.
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Materials and Methods |
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Metal Particles
Particles of pure titanium (Johnson Matthey Chemicals, Ward Hill, Massachusetts), one to three micrometers in diameter, were washed three times with sterile phosphate-buffered saline solution and were sterilized in an autoclave. For use in monocyte cultures, the particles were resuspended in sterile phosphate-buffered saline solution at a concentration of 6 x 108 particles per milliliter. The concentration and size of the particles were determined by analysis with a Channelizer (model ZM; Coulter Electronics, Hialeah, Florida). More than 90 per cent of the particles were less than five micrometers in diameter. Particle suspensions were shown to be free of endotoxin by Limulus assay (BioWhittaker, Walkersville, Maryland).
Isolation and Culture of Monocytes
Thirty milliliters of fresh venous blood from five healthy volunteers or sixty milliliters of soft-spin buffy coat from three American Red Cross donors was diluted one to one with phosphate-buffered saline solution and was underlaid with a one-third volume of warm Lymphoprep solution (NycoMed AS, Oslo, Norway). Samples were centrifuged to separate monocytes and lymphocytes from the remaining blood elements. The middle layer, containing lymphocytes and monocytes, was aspirated and transferred to a fresh tube, washed twice with sterile phosphate-buffered saline solution, and resuspended at a concentration of two to five million cells per milliliter in medium (RPMI-1640; Sigma Chemical, St. Louis, Missouri) containing 10 per cent fetal bovine serum and 5 per cent tumor media additions (2000 units of penicillin per milliliter, 200 micrograms of streptomycin per milliliter, two-millimolar L-glutamine, one-millimolar minimum essential medium non-essential amino acids, and thirteen-millimolar sodium bicarbonate) (DIFCO Labs, Detroit, Michigan, and Gibco BRL Life Technologies, Grand Island, New York). Thirty milliliters of whole blood consistently yielded a mixed population of twenty to thirty million lymphoctyes and monocytes, whereas sixty milliliters of the buffy-coat layer provided 450 to 650 million cells.
Monocytes were separated from lymphocytes by adherence in a thirty-milliliter tissue-culture flask for one hour at 37 degrees Celsius and 7.5 per cent CO2. Lymphocytes were washed from the adherent monocytes twice with serum-free medium. The monocytes were incubated for fifteen minutes at 37 degrees Celsius in three milliliters of phosphate-buffered saline solution containing one-millimolar EDTA and 10 per cent fetal bovine serum and then were gently scraped from the flask. The monocytes were washed twice with a solution of sterile medium and phosphate-buffered saline solution and were resuspended in a solution of medium and fetal bovine serum. In some experiments, monocytes were plated in serum-free medium, to assess the effect of serum factors on phagocytosis. Approximately 10 to 15 per cent of the initial population of monocytes and lymphocytes was composed of monocytes.
The isolated monocytes were plated at one million cells (buffy-coat layer from donated blood) or 100,000 cells (whole blood from volunteers) per milliliter of medium containing 10 per cent fetal bovine serum and were immediately treated with titanium particles as described. For experiments performed to examine the effects of titanium particles on the levels of protein, cells in one milliliter of medium were placed in twenty-four-well culture plates with the wells having a diameter of sixteen millimeters. For the examination of the effects of titanium particles on the synthesis of mRNA, the cells were added in ten milliliters of medium to a cell-culture dish with a diameter of 100 millimeters.
Histological Analysis of Monocyte Cultures
Cells were examined with light microscopy and the trypan-blue exclusion method to verify their viability. For histological analysis, monocytes were treated with 4 x 105 titanium particles per milliliter of medium for eighteen hours, rinsed with phosphate-buffered saline solution, and subjected to phase-contrast microscopy. In order to confirm phagocytosis, the monocytes were treated with titanium particles in twenty-four-well tissue-culture plates for eighteen hours, fixed in 4 per cent paraformaldehyde, and embedded in epoxy resin according to a previously described protocol9. The cells were removed from the culture dish and sections, 2.5 micrometers thick, were cut and analyzed.
Assay for Cytokines
After the medium had been incubated with titanium particles, conditioned medium was obtained and the titanium particles were removed from the suspension by centrifugation at 9000 revolutions per minute at 4 degrees Celsius. The supernatants were stored at -70 degrees Celsius and were thawed once for the measurement of the cytokine levels by enzyme-linked immunosorbent assay. For quantification of the levels of cell-associated cytokines, the culture medium was removed and the cultures were rinsed three times with phosphate-buffered saline solution. One milliliter of phosphate-buffered saline solution was then added to the cultures, which were subjected to three freeze-thaw cycles (between 4 and -70 degrees Celsius.)
The levels of IL-6 and TNF- were measured with use of commercially available enzyme-linked immunosorbent assay kits (Biosource International, Camarillo, California, and Pharmingen, San Diego, California). The so-called sandwich enzyme-linked immunosorbent assay kits consisted of an anti-cytokine-detecting antibody bound to the plate, followed by a second biotinylated anti-cytokine antibody. Avidin-peroxidase enzyme and ABTS chromagen (Sigma Chemical) were added, and the change in color was measured with spectroscopy at 450 or 405 nanometers, respectively, on an automatic microELISA plate reader (model MRS 80; Dynatech Labs, Alexandria, Virginia). Optical densitometry results were then compared with standard concentrations of the appropriate cytokine (Biosource International).
Isolation of RNA
For the isolation of mRNA, 1 x 106 monocytes were plated in a tissue-culture dish (diameter, 100 millimeters) and treated with medium containing serum and varying concentrations of titanium particles. The conditioned medium was aspirated from the monocyte cultures at specified times, and the plates were washed with sterile phosphate-buffered saline solution. The cells were immediately placed on ice, and six milliliters of RNAzol reagent (Tel-Test, Friendswood, Texas) or TRIzol reagent (Gibco BRL Life Technologies) was added to the cell population. The cells were gently scraped from the plates and transferred to conical tubes, a one-fifth volume of chloroform was added, and the tubes were centrifuged for fifteen minutes at 12,000 times gravity (4 degrees Celsius). The aqueous phase was transferred to a new tube and mixed with one volume of isopropanol. After centrifugation, the supernatant was discarded and the RNA pellet was washed with 75 per cent ethanol and then dried under vacuum. The RNA pellet was resuspended in 0.4 milliliter of ten-millimolar Tris and one-millimolar EDTA (pH 8.0), and a second precipitation was performed with a one-tenth volume of 0.3-molar sodium acetate and two and one-half volumes of 95 per cent ethanol. The concentration of total cellular RNA was determined with use of ultraviolet spectroscopy at 260 and 280 nanometers. A population of ten million monocytes yielded approximately five to fifteen micrograms of total RNA.
Northern Hybridization
Five-microgram aliquots of total cellular RNA were separated on a 1.2 per cent agarose gel under denaturing conditions (17.5 per cent formaldehyde). RNA was transferred to nylon membranes (GeneScreen Plus; Biotechnology Systems, Boston, Massachusetts) with a ten-times-standard concentration of saline-sodium citrate solution (three-molar sodium chloride and 0.15-molar sodium citrate) overnight and baked at 80 degrees Celsius for two hours. The membranes were then placed in hybridization solution (a six-times-standard concentration of saline-sodium citrate solution, a five-times-standard concentration of Denhardt reagent, 10 per cent dextran sulfate, 1 per cent sodium dodecyl sulfate, and denatured salmon sperm DNA [100 micrograms per milliliter]) (5'-3' Incorporated, Boulder, Colorado) for one hour at 65 degrees Celsius. 32P-labeled complementary DNA (cDNA) probes specific for TNF- or IL-6 were added at a concentration of 1 x 106 counts per milliliter, and hybridization was performed at 65 degrees Celsius for twelve hours. Non-specific binding was removed by four sequential washes at 65 degrees Celsius for ten minutes each with a four-times-standard concentration of saline-sodium citrate solution with 1 per cent sodium dodecyl sulfate, a two-times-standard concentration of saline-sodium citrate solution with 1 per cent sodium dodecyl sulfate, a 0.5-times-standard concentration of saline-sodium citrate solution with 0.1 per cent sodium dodecyl sulfate, and a 0.1-times-standard concentration of saline-sodium citrate solution with 0.1 per cent sodium dodecyl sulfate. Filters were exposed to XAR 5 film (Eastman Kodak, Rochester, New York) at -70 degrees Celsius.
Autoradiographs of the Northern blots were scanned with use of a densitometer (Molecular Dynamics, Sunnyvale, California), and the volume of the signal was quantified with use of Image Quant Software (Molecular Dynamics).
The TNF- cDNA clone was provided by Dr. Chris Ritchlin (Department of Immunology, Rochester General Hospital, Rochester, New York). The TNF- fragment that was randomly primed for Northern blot was an 1108-base-pair PstI restriction fragment from a human cDNA clone (ATCC 39894; American Type Culture Collection [ATCC], Rockville, Maryland). The human IL-6 cDNA clone was obtained from American Type Culture Collection, and the fragment used for random priming was a 1160-base-pair EcoR1 fragment (ATCC 67153). A cDNA sequence specific for the human 28S ribosome was obtained from Ambion (Austin, Texas). 32P-deoxycytidine-5'-triphosphate-labeled probes were synthesized with use of a commercially available kit (Random Primers Labeling System; Gibco BRL Life Technologies).
Pharmacological Agents
All pharmacological agents were purchased from Sigma Chemical. Lipopolysaccharide was diluted in sterile medium at a final concentration of 100 nanograms per milliliter. Actinomycin D and cycloheximide were each suspended in water, diluted in medium at a final concentration of ten micrograms per milliliter, and sterile-filtered before use in the cell-culture experiments.
Statistical Analysis
Statistical analysis was performed with use of a paired Student t test.
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Results |
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Particle Phagocytosis
The titanium-treated monocyte cultures were examined with phase-contrast microscopy (Fig. 1-A). Although phagocytosis is suggested by the association of titanium particles with monocytes, the findings can be due to a concentration of titanium particles at the cell membrane. To confirm phagocytosis, 2.5-micrometer plastic-embedded sections from titanium-treated monocyte cultures were also analyzed (Fig. 1-B). Because the thickness of the sections was substantially less than the diameter of the monocytes, the titanium particles located within the cell represented phagocytosed particles. Cultured human monocytes readily phagocytosed titanium particles in all of the cultures examined. Phagocytosis was independent of serum proteins, as it occurred in both serum-free and serum-containing medium. Phagocytosis did not result in the death of cells, as determined by trypan-blue exclusion (data not shown). In cultures that were examined as long as eighteen hours after treatment with titanium particles, cell viability was greater than 95 per cent and was similar in control and titanium-treated cultures.
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Production of Cytokines in Cell Supernatants
The effect of titanium particles on the synthesis of TNF- (Fig. 2-A) and IL-6 (Fig. 2-B) was examined eighteen to twenty-two hours after plating and exposure to titanium particles. Titanium particles stimulated a dose-dependent increase in the production of TNF- and IL-6 in all isolated human peripheral blood monocytes. The response was qualitatively similar in all of the cultures, including those of whole blood from volunteers, cultured at 100,000 cells per milliliter, as well as those of the buffy-coat layer obtained from donated blood, cultured at 1 x 106 cells per milliliter. The effects were observed at concentrations as low as 6 x 104 titanium particles per milliliter, which demonstrates a wide range of stimulatory activity. The release of TNF- into the medium was the greatest at a concentration of 6 x 105 to 6 x 106 particles per milliliter; generally, a higher concentration of titanium particles was needed for a maximum response in the monocytes from the buffy-coat layer (Fig. 2-A). The fact that more particles were needed to stimulate the buffy-coat monocytes may be due to the tenfold increase in cells in these cultures, which effectively reduces the number of phagocytosable particles per cell. However, the similarity in response validates the utilization of buffy-coat monocytes and permits experiments, such as the isolation of mRNA, that require an increased number of monocytes. The level of TNF- was fifty to 1000 times that of the control levels; the control levels in the cultures of the buffy-coat monocytes were within the range observed in the cultures of the whole-blood monocytes.
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Monocytes responded to titanium particles with a dose-dependent increase in the synthesis of IL-6 that paralleled that of TNF-
, except at the highest concentration of titanium particles, which resulted in a decrease in the level of IL-6 (Fig. 2-B). Although there was a tendency for a slight decrease in the level of TNF- at the highest concentration of titanium particles, this effect was more pronounced with IL-6: the level was the same as the basal level of secretion. The biphasic response is not due to cellular toxicity, as a similar effect was not observed for TNF- and the cells were viable at all concentrations of titanium particles, according to trypan-blue exclusion tests. Although stimulation with titanium particles produced levels of IL-6 that were greater than those of TNF-, the levels of IL-6 were only two to five times that of the control levels. These findings were due to the relatively high basal levels of IL-6 in the monocyte cultures, which are consistent with the high basal levels of IL-6 that have been observed in other studies15.
The kinetics of the production of TNF- and IL-6 were examined in cultures of human peripheral blood monocytes treated with the maximally stimulatory concentration of titanium particles (Fig. 3). The levels of TNF- in the conditioned medium increased from an undetectable level (less than fifteen picograms per milliliter) to 192 picograms per milliliter by two hours, with the greatest level (1169 picograms per milliliter) at eight hours. The level remained in the maximum range for twenty-four hours and then steadily declined over a seventy-two-hour incubation. The level of IL-6 in the conditioned medium increased to 124 picograms per milliliter after four and one-half hours of incubation and was the highest (657 picograms per milliliter) at sixteen hours. The level of IL-6 remained elevated after incubation for seventy-two hours.
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To demonstrate the absence of a soluble mediator of monocyte activation, such as lipopolysaccharide or metal ions, culture medium was conditioned with titanium particles (6 x 106 per milliliter) for six hours, and the particles were removed by centrifugation. Monocyte cultures were incubated in the conditioned medium for eighteen hours, and the results were compared with those for control cultures and titanium-treated cultures; there was no difference between the control and the conditioned medium-treated cultures with regard to the levels of TNF-
or IL-6. In contrast, cultures treated with titanium particles had elevated levels of both TNF- and IL-6, findings that were consistent with the previously described results (data not shown). These experiments confirmed the role of particles of debris in the stimulation of the cytokine response.
Levels of Stored Cytokines in Peripheral Blood Monocytes
The rapid increase in the levels of TNF- and IL-6 suggests that their secretion may be related to a release from intracellular stores and not to the de novo synthesis of these proteins. In order to address this issue, the levels of intracellular and extracellular cytokines were measured in control and titanium-treated cultures immediately after stimulation and at eighteen hours (Figs. 4-A and 4-B). The data demonstrated very low levels of immunoreactive TNF- and IL-6 within the cell immediately after stimulation, indicating that protein synthesis is needed for the secretion of these cytokines. Although a toxic effect could result in the release of intracellular stores, the need for new protein synthesis suggests activation of biochemical pathways resulting in the production and secretion of cytokines.
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At eighteen hours, the intracellular content of TNF-
and IL-6 was slightly higher in the titanium-treated cultures than in the controls, but the difference was substantially greater in the extracellular (medium) fraction. This suggests that newly synthesized TNF- and IL-6 are rapidly secreted and intracellular storage pools remain small. The increase in the levels of TNF- and IL-6 in the control cultures at eighteen hours is probably related either to basal levels of synthesis or, more likely, to slight activation of the monocytes in response to cell culture.
Transcriptional and Translational Regulation of Cytokine Production
In order to evaluate the role of protein and mRNA synthesis in the secretion of TNF- and IL-6 in peripheral blood monocytes stimulated with titanium particles, the cultures were treated with cycloheximide, a potent inhibitor of protein synthesis, and actinomycin D, an inhibitor of RNA synthesis. The induction by titanium of synthesis of TNF- was inhibited in a dose-dependent manner in monocyte cultures treated with a maximally stimulatory concentration of titanium particles for eighteen hours. One hundred per cent inhibition was observed with each inhibitor at a concentration of five micrograms per milliliter (Fig. 5). Similar results were obtained with IL-6 (data not shown). The results demonstrate that the synthesis of both protein and mRNA is necessary for titanium-induced production of TNF- and IL-6 in human peripheral blood monocytes.
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To evaluate the role of mRNA synthesis in the production of cytokines further, total RNA was isolated from control and titanium-treated cultures. Time-course experiments with 6 x 106 titanium particles demonstrated rapid increases in the levels of TNF-
and IL-6 mRNA (Figs. 6-A and 6-B). The level of TNF- mRNA was greatest at ninety minutes and then decreased to the control level at six hours. In contrast, the level of IL-6 mRNA increased gradually, with the greatest level at three hours. The maximally stimulatory concentration of titanium particles (6 x 106) resulted in a level of TNF- mRNA that was five times the control level and a level of IL-6 mRNA that was six to twelve times the control level. To ensure that the increases in the levels of mRNA were not secondary to a non-specific effect of isolation and culture, control levels of mRNA were obtained for each time-point. Although there was a slight increase in the level of mRNA in the control cultures, titanium reproducibly stimulated mRNA synthesis in human monocyte cultures. The slight increase in the levels of TNF- and IL-6 mRNA in the control cultures, which paralleled the increase observed in the titanium-treated cultures, further suggests that culture of peripheral blood monocytes may result in slight activation of these cells.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The present study demonstrated that when human peripheral blood monocytes are stimulated by titanium particles, they secrete TNF-
and IL-6. The response required de novo synthesis of both TNF- and IL-6 mRNA and protein and was rapid, with an increase in the level of TNF- mRNA at thirty minutes and of IL-6 mRNA at ninety minutes. The level of TNF- protein was greatest at eight hours, whereas the level of IL-6 protein was greatest at sixteen hours. This is consistent with the later induction of the synthesis of IL-6 mRNA in peripheral blood monocytes. The response occurred over a broad range of concentrations of titanium particles, but it was dose-dependent, with the greatest response at a concentration of 6 x 105 to 6 x 106 titanium particles per milliliter.
Peripheral blood monocytes are precursors of tissue macrophages and are key regulators of the inflammatory response20,25. Activation of these cells occurs with phagocytosis of foreign material and results in the secretion of cytokines, including IL-6 and TNF-, which drive the inflammatory response but also are involved in the stimulation of osteoclastic bone resorption5,11,13,26,27. Lipopolysaccharide, through effects on the cell membrane, also activates monocytes and macrophages and the release of cytokines from these cells. The effect of titanium particles in the present study was not related to contamination of the titanium particles with lipopolysaccharide or to the effects of soluble metal ions, as medium conditioned with titanium particles did not stimulate the release of cytokines. Instead, activation was related to phagocytosis of titanium particles, with the subsequent stimulation of intracellular pathways leading to increased synthesis and secretion of TNF- and IL-6.
The secretion of TNF- and IL-6 increased in the monocytes from all of the specimens of whole blood as well as from all of the buffy-coat layers from donated blood. The responses of the two cell preparations were qualitatively similar, although a greater number of particles was necessary to produce maximum effects in the monocytes from the buffy-coat layer. This may be because of the tenfold increase in the number of cells in these cultures, which effectively reduced the number of phagocytosable particles per cell. Although there was a tendency for a slight decrease in the level of TNF- at the greatest concentration of titanium particles, this effect was much more pronounced with regard to the level of IL-6, which returned to the basal level of secretion at the greatest concentration of particles in each of the cultures of monocytes from whole blood. The qualitative differences in the synthesis of TNF- and IL-6 mRNA and protein suggest that the effect may result from the stimulation of separate but perhaps interrelated biochemical pathways.
Although previous studies examining the synthesis of TNF- in lipopolysaccharide (endotoxin)-stimulated macrophages have suggested that the regulation of this synthesis in monocytes involves complex transcriptional and post-transcriptional mechanisms5,11,13,26,27, the mechanism of response to particles of debris has not previously been elucidated. In titanium-treated monocyte cultures, the level of TNF- mRNA was five times the control level, whereas the level of TNF- in the medium was fifty to 1000 times the control level. These observations are consistent with those in other investigations with lipopolysaccharide-stimulated monocytes, which have demonstrated a threefold increase in the level of TNF- mRNA compared with a 10,000-fold increase in the level of TNF- protein5,13. Low levels of TNF- mRNA may account for relatively high levels of protein synthesis, but the converse is also true. We observed negligible synthesis of TNF- protein in unstimulated control cultures, despite the presence of relatively high levels of TNF- mRNA.
These observations suggest that important post-transcriptional regulatory mechanisms for the release of TNF- are operational in titanium-treated monocyte cultures. Previous work with lipopolysaccharide-stimulated monocytes has identified several putative post-transcriptional regulatory steps involved in the secretion of TNF-. These steps involve processing and stability of mRNA, packaging of TNF- into microsomes for later secretion, and anchoring of the TNF- precursor to the cell membrane, which requires proteolytic cleavage for secretion5,11,13,27. These complicated control mechanisms may act separately or in combination to limit the secretion of cytokines under normal conditions, even in the presence of relatively high basal levels of mRNA.
The increase in the level of TNF- protein was several orders of magnitude greater than the increase in mRNA levels; in contrast, the level of IL-6 appeared to be more directly associated with changes in the synthesis of mRNA. This suggests that IL-6 is regulated primarily by a transcriptional mechanism, whereas TNF- is regulated by both transcriptional and post-transcriptional mechanisms. However, secretion of both TNF- and IL-6 depends on de novo synthesis of mRNA and protein, as production of both cytokines was blocked by actinomycin D and cycloheximide. These latter findings were supported by experiments demonstrating low intracellular stores of TNF- and IL-6 in both stimulated and unstimulated monocyte cultures.
The present investigation is the first, to our knowledge, to demonstrate stimulation of TNF- and IL-6 by titanium particles in isolated peripheral blood monocytes. The concentrations of titanium particles used in the present investigation were well within the range observed in vivo (1 x 109 particles per gram of tissue) and support the physiological relevance of the present findings23. The rapid stimulation of the production of TNF- and IL-6 was followed by a steady decrease in the levels of the cytokines after twenty-four hours and was probably due to the metabolism of these proteins in the absence of any additional synthesis. This is consistent with the transient nature of the increase in the levels of TNF- and IL-6 mRNA after stimulation by titanium particles. Although these findings were reproducible, the comparison of an in vivo model with in vitro events is uncertain, particularly considering the potential role of the other interacting cell types involved in the more complicated periprosthetic tissue response.
Although human peripheral blood monocytes have been used extensively in previous hematological investigations21,28,29, there has been considerably less experience with these cells for the study of the effects of particles of debris. These cells have advantages compared with previously used cell lines and animal models, as none of the effects can be attributed to differences in species or alterations in cell phenotype due to transformation of a cell line.
In this study, peripheral blood monocytes were treated immediately after plating, as preliminary studies demonstrated that cells placed in overnight culture before stimulation showed a blunted cytokine response, suggesting that prolonged culture conditions may alter the responsiveness of peripheral blood monocytes. Peripheral blood monocytes in cell culture produced basal levels of cytokines and demonstrated a time-dependent response of mRNA levels in control cultures that appears to parallel the changes in the titanium-treated cultures. This suggests that the process of plating the cells in culture may cause a mild activation of the cells with a subsequent increase in mRNA, but we believe that this finding does not alter the validity of the model or the present experiments, given the considerable effects that were observed in the titanium-treated cultures. The experiments performed in the present study demonstrate that peripheral blood monocytes from all individuals tested responded to titanium particles by increasing the synthesis of cytokines.
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Footnotes |
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*No benefits in any form have been received or will be received from a commercial party related directly or indirectly to the subject of this article. Funds were received in total or partial support of the research or clinical study presented in this article. The funding sources were the Career Development Award (R. J. O'K.) and the Resident Research Award (T. A. B.) from the Orthopaedic Research and Education Foundation.
Department of Orthopaedics, Box 665, and Department of Immunology (R. J. L.), University of Rochester School of Medicine and Dentistry, 601 Elmwood Avenue, Rochester, New York 14642.
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References |
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- Algan, S. M., and |and |Horowitz, S. M.: Role of tumor necrosis factor in particulate induced bone resorption associated with aseptic loosening of prosthetic joint replacements. Trans. Orthop. Res. Soc., 20: 285, 1995.
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Appel, A. M.; Sowder, W. G.; Siverhus, S. W.; Hopson, C. N.; and |and |Herman, J. H.: Prosthesis-associated pseudomembrane-induced bone resorption. British J. Rheumatol., 29: 32-36, 1990.
[Abstract/Free Full Text] - Athanasou, N. A.; Quinn, J.; and |and |Bulstrode, C. J. K.: Resorption of bone by inflammatory cells derived from the joint capsule of hip arthroplasties. J. Bone and Joint Surg., 74-B(1): 57-62, 1992.
- Bertolini, D. R.; Nedwin, G. E.; Bringman, T. S.; Smith, D. D.; and |and |Mundy, G. R.: Stimulation of bone resorption and inhibition of bone formation in vitro by human tumour necrosis factors. Nature, 319: 516-518, 1986.[Medline]
- Beutler, B.: Tumor Necrosis Factors: the Molecules and Their Emerging Role in Medicine. New York, Raven Press, 1992.
- Boynton, E.; Waddell, J. P.; Morton, J.; and |and |Gardiner, G. W.: Aseptic loosening in total hip implants: the role of polyethylene wear debris. Canadian J. Surg., 34: 599-605, 1991.[Medline]
- Buly, R. L.; Huo, M. H.; Salvati, E.; Brien, W.; and |and |Bansal, M.: Titanium wear debris in failed cemented total hip arthroplasty. An analysis of 71 cases. J. Arthroplasty, 7: 315-323, 1992.[Medline]
- Chiba, J.; Rubash, H. E.; Kim, K. J.; and |and |Iwaki, Y.: The characterization of cytokines in the interface tissue obtained from failed cementless total hip arthroplasty with and without femoral osteolysis. Clin. Orthop., 300: 304-312, 1994.
- di Sant'Agnese, P. A., and |and |De Mesy-Jensen, K. L.: Diagnostic electron microscopy on reembedded ("popped off") areas of large spurr epoxy sections. Ultrastruct. Pathol., 6: 247-253, 1984.[Medline]
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Friedman, R. J.; Black, J.; Galante, J. O.; Jacobs, J. J.; and |and |Skinner, H. B.: Current concepts in orthopaedic biomaterials and implant fixation. J. Bone and Joint Surg., 75-A: 1086-1109, July 1993.
[Free Full Text] -
Gearing, A. J.; Beckett, P.; Christodoulou, M.; Churchill, M.; Clements, J.; Davidson, A. H.; Drummond, A. H.; Galloway, W. A.; Gilbert, R.; Gordon, J. L.; Leber, T. M.; Mangan, M.; Miller, K.; Nayee, P.; Owen, K.; Patel, S.; Thomas, W.; Wells, G.; Wood, L. M.; and |and |Woolley, K.: Processing of tumour necrosis factor- precursor by metalloproteinases [letter]. Nature, 370: 555-557, 1994.[Medline]
- Glant, T. T., and |and |Jacobs, J. J.: Response of three murine macrophage populations to particulate debris: bone resorption in organ cultures. J. Orthop. Res., 12: 720-731, 1994.[Medline]
-
Han, J.; Brown, T.; and |and |Beutler, B.: Endotoxin-responsive sequences control cachectin/tumor necrosis factor biosynthesis at the translational level. J. Exper. Med., 171: 465-475, 1990.
[Abstract/Free Full Text] - Harada, Y.; Brown, S.; Merritt, K.; Wang, J. T.; Doppalappudi, V. A.; Willis, A. A.; Jasty, M.; Harris, W. H.; and |and |Goldring, S. R.: Effects of metal particles and their corrosion products on human monocyte/macrophages in vitro. Trans. Orthop. Res. Soc., 20: 776, 1995.
-
Haynes, D. R.; Rogers, S. D.; Hay, S.; Pearcy, M. J.; and |and |Howie, D. W.: The differences in toxicity and release of bone-resorbing mediators induced by titanium and cobalt-chromium-alloy wear particles. J. Bone and Joint Surg., 75-A: 825-834, June 1993.
[Abstract/Free Full Text] -
Horowitz, S. M.; Doty, S. B.; Lane, J. M.; and |and |Burstein, A. H.: Studies of the mechanism by which the mechanical failure of polymethylmethacrylate leads to bone resorption. J. Bone and Joint Surg., 75-A: 802-813, June 1993.
[Abstract/Free Full Text] - James, R. E.; Maloney, W. J.; and |and |Smith, R. L.: The morphologic and metabolic response of isolated human macrophages to retrieved titanium alloy particles. Trans. Orthop. Res. Soc., 20: 775, 1995.
-
Jiranek, W. A.; Machado, M.; Jasty, M.; Jevsevar, D.; Wolfe, H. J.; Goldring, S. R.; Goldberg, M. J.; and |and |Harris, W. H.: Production of cytokines around loosened cemented acetabular components. Analysis with immunohistochemical techniques and in situ hybridization. J. Bone and Joint Surg., 75-A: 863-879, June 1993.
[Abstract/Free Full Text] -
Kim, K. J.; Chiba, J.; and |and |Rubash, H. E.: In vivo and in vitro analysis of membranes from hip prostheses inserted without cement. J. Bone and Joint Surg., 76-A: 172-180, Feb. 1994.
[Abstract/Free Full Text] - Krakauer, T., and |and |Oppenheim, J. J.: IL-1 and tumor necrosis factor-alpha each up-regulate both the expression of IFN-gamma receptors and enhance IFN-gamma-induced HLA-DR expression on human monocytes and a human monocytic cell line (THP-1). J. Immunol., 150: 1205-1211, 1993.[Abstract]
- Liebler, J. M.; Kunkel, S. L.; Burdick, M. D.; Standiford, T. J.; Rolfe, M. W.; and |and |Strieter, R. M.: Production of IL-8 and monocyte chemotactic peptide-1 by peripheral blood monocytes. Disparate responses to phytohemagglutinin and lipopolysaccharide. J. Immunol., 152: 241-249, 1994.[Abstract]
-
Maloney, W. J.; Jasty, M.; Harris, W. H.; Galante, J. O.; and |and |Callaghan, J. J.: Endosteal erosion in association with stable uncemented femoral components. J. Bone and Joint Surg., 72-A: 1025-1034, Aug. 1990.
[Abstract/Free Full Text] -
Margevicius, K. J.; Bauer, T. W.; McMahon, J. T.; Brown, S. A.; and |and |Merritt, K.: Isolation and characterization of debris in membranes around total joint prostheses. J. Bone and Joint Surg., 76-A: 1664-1675, Nov. 1994.
[Abstract/Free Full Text] -
Martell, J. M.; Pierson, R. H., III; Jacobs, J. J.; Rosenberg, A. G.; Maley, M.; and |and |Galante, J. O.: Primary total hip reconstruction with a titanium fiber-coated prosthesis inserted without cement. J. Bone and Joint Surg., 75-A: 554-571, April 1993.
[Abstract/Free Full Text] - Parslow, T. G.: The phagocytes: neutrophils and macrophages. In Basic and Clinical Immunology, edited by D. P. Stites, A. I. Terr, and T. G. Parslow. Ed. 8, pp. 9-21. Norwalk, Connecticut, Appleton and Lange, 1994.
-
Shakhov, A. N.; Collart, M. A.; Vassalli, P.; Nedospasov, S. A.; and |and |Jongeneel, C. V.: kB-type enhancers are involved in lipopolysaccharide-mediated transcriptional activation of the tumor necrosis factor- gene in primary macrophages. J. Exper. Med., 171: 35-47, 1990.
[Abstract/Free Full Text] - Sung, S. J.; Walters, J. A.; Hudson, J.; and |and |Gimble, J. M.: Tumor necrosis factor-alpha mRNA accumulation in human myelomonocytic cell lines. Role of transcriptional regulation by DNA sequence motifs and mRNA stabilization. J. Immunol., 147: 2047-2054, 1991.[Abstract]
- Terao, I.; Hashimoto, S.; and |and |Horie, T.: Effect of GM-CSF on TNF-alpha and IL-1-beta production by alveolar macrophages and peripheral blood monocytes from patients with sarcoidosis. Internat. Arch. Allergy and Immunol., 102: 242-248, 1993.
- Wangoo, A.; Haynes, A. R.; Sutcliffe, S. P.; Sorooshian, M.; and |and |Shaw, R. J.: Modulation of platelet-derived growth factor B mRNA abundance in macrophages by colchicine and dibutyryl-cAMP. Molec. Pharmacol., 42: 584-589, 1992.[Abstract]
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