The Journal of Bone & Joint Surgery

The Journal of Bone & Joint Surgery, Volume 89, Issue 5

Scientific Articles | May 01, 2007

Inhibition of the PI3K-Akt Signaling Pathway Reduces Tumor Necrosis Factor-α Production in Response to Titanium Particles in Vitro

Matthew V. Smith, MD¹; Michael J. Lee, MD¹; Andrew S. Islam, MD¹; Jacqueline L. Rohrer, BS²; Victor M. Goldberg, MD¹; Michelle A. Beidelschies, BS³; Edward M. Greenfield, PhD³

¹ Department of Orthopaedics, Case Western Reserve University/University Hospitals of Cleveland, 11100 Euclid Avenue, Cleveland, OH 44106. E-mail address for M.V. Smith: mvsmith23@gmail.com
² 4675 Arbour Green Drive, Akron, OH 44333
³ Departments of Physiology and Biophysics (M.A.B.) and Orthopaedics (E.M.G.), Case Medical Center, Case Western Reserve University, Biomedical Research Building, Room 331, 2109 Adelbert Road, Cleveland, OH 44106. E-mail address for M.A. Beidelschies: mai6@case.edu. E-mail address for E.M. Greenfield: emg3@po.cwru.edu

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Disclosure: In support of their research for or preparation of this work, one or more of the authors received, in any one year, outside funding or grants in excess of $10,000 from the National Institutes of Health (research grant RO1 AR43769 and training grant AR07505) and three authors were supported by Allen Fellowships. Neither they nor a member of their immediate families received payments or other benefits or a commitment or agreement to provide such benefits from a commercial entity. No commercial entity paid or directed, or agreed to pay or direct, any benefits to any research fund, foundation, division, center, clinical practice, or other charitable or nonprofit organization with which the authors, or a member of their immediate families, are affiliated or associated.

Investigation performed at the Departments of Orthopaedics, Physiology and Biophysics, and Pathology, Case Western Reserve University and University Hospitals, Cleveland, Ohio

The Journal of Bone and Joint Surgery, Incorporated

J Bone Joint Surg Am, 2007 May 01;89(5):1019-1027. doi: 10.2106/JBJS.F.00615

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Abstract

Background: Wear debris contributes to implant loosening after total joint arthroplasty, and few advances have been made in our ability to inhibit the biological response to wear particles. Bacterial endotoxins augment the effects of wear particles in vitro and in vivo. The cytokine, tumor necrosis factor-a (TNF-a), is produced by macrophages in response to bacterial endotoxins and wear particles, and it increases osteoclast activity resulting in bone resorption and implant loosening. The phosphoinositol-3-kinase (PI3K)-Akt intracellular signal transduction pathway contributes to cytokine production in response to soluble endotoxin. We investigated the role of the PI3K-Akt pathway in the production of TNF-a in response to wear particles with adherent endotoxin and so-called endotoxin-free wear particles.

Methods: Cultured RAW264.7 murine macrophages were incubated with titanium particles with adherent endotoxin or with endotoxin-free titanium particles in the presence and absence of specific inhibitors of PI3K (LY294002) or Akt (SH-5). Akt activation was assessed with use of Western blot. TNF-a production was measured with use of enzyme-linked immunosorbent assay. Cytotoxicity was determined by measuring lactic dehydrogenase release.

Results: Titanium particles with adherent endotoxin increased Akt activation, whereas endotoxin-free titanium particles did not. The PI3K inhibitor reduced TNF-a production by 70% in response to titanium with adherent endotoxin without increasing cytotoxicity. Similarly, the Akt inhibitor reduced TNF-a production by 83% in response to titanium particles with adherent endotoxin without increasing cytotoxicity. High concentrations of endotoxin-free titanium particles resulted in a small delayed increase in TNF-a production that was completely blocked by the PI3K inhibitor.

Conclusions: Inhibition of the PI3K-Akt pathway reduces macrophage TNF-a production in response to titanium particles with adherent endotoxin and endotoxin-free particles in vitro.

Clinical Relevance: In vivo studies are needed as these results suggest a possible pharmacological target to reduce wear particle-induced osteolysis and subsequent implant loosening.

Figures in this Article

Joint replacement surgery is an effective way to treat patients with severe arthritis. Unfortunately, many joint replacements fail over time because of aseptic loosening. Loosening in the absence of clinical or microbiological signs of infection is, in part, caused by wear debris generated from the implants¹. Advances in biomaterials and surgical technique have been developed in an effort to reduce wear debris¹. The advent of improved bearing surfaces, such as cross-linked polyethylene, will likely reduce the rate of particle generation. However, this approach alone will be unlikely to eliminate aseptic loosening since it does not completely block particle generation from the bearing surfaces²; leads to the production of smaller, more biologically active particles³; and will likely have little effect on particles generated from third-body wear, backside wear, cement degradation, corrosion, fretting, malalignment, wear of the Morse tapers, or delamination of hydroxyapatite coatings¹. Understanding the biological effects of wear debris may provide for pharmacological treatments to reduce the prevalence of implant loosening.

Wear particles activate macrophages to release proinflammatory cytokines, such as interleukin-1 (IL-1), IL-6, and tumor necrosis factor-a (TNF-a)⁴. However, wear particles themselves may not be the sole stimulus of cytokine production in aseptic loosening. Rather, bacteria-derived endotoxins that are adherent to the wear particles may provide an increased inflammatory stimulus^5-7. Lipopolysaccharide from gram-negative bacteria as well as lipoteichoic acid and peptidoglycan from gram-positive bacteria induce cytokine production and have similar biological effects^8,9. There is evidence that these endotoxins may be important for the biological activity of wear debris. First, removal of endotoxin from titanium particles results in a substantial reduction of TNF-a, IL-1, IL-6, osteoclast differentiation, and osteolysis induced by titanium particles⁴. Adding back adherent lipopolysaccharide to the so-called endotoxin-free particles restores their ability to induce these cytokines⁴. In addition, the biological effects of the titanium particles depend on Toll-like receptor-4 (TLR-4), which is the primary receptor for lipopolysaccharide⁴.

There is also clinical evidence that bacterial endotoxins may be important in the development of aseptic loosening. A study of >20,000 hip replacements in the Norwegian Arthroplasty Register showed that systemic antibiotics and antibiotics in cement reduced the incidence of aseptic loosening¹⁰. Also, a gram-positive bacterial biofilm has been found on many implants retrieved from patients with no clinical evidence of infection⁵. This biofilm may be a source of local endotoxin. An additional source of local endotoxin may be the implants themselves, as adherent endotoxin (twelve to fifteen units of endotoxin per square meter) has been found on titanium discs sterilized, processed, and packaged by a major orthopaedic implant manufacturer in the same manner as their commercially available joint replacements¹¹. Additionally, endotoxin may accumulate from systemic sources as suggested by the fact that endotoxin-free titanium and polyethylene particles accrue endotoxin in the first seven days after implantation on mouse calvaria¹². Potential sources of accumulated endotoxin include minor infections, gut flora, and, in humans, dental procedures¹³. Accumulation from these sources may explain why endotoxin exists in periprosthetic tissue from patients with aseptic loosening and inflammatory arthritis and why peptidoglycan exists in synovial tissue of patients with either rheumatoid arthritis or osteoarthritis^14,15. The clinical relevance of these potential local and systemic sources of endotoxin requires additional investigation.

The cell-signaling mechanisms by which wear particles induce macrophages to generate cytokines are incompletely understood. However, there is a substantial body of literature examining the macrophage response to lipopolysaccharide. Multiple signaling pathways, including the phosphoinositol-3-kinase (PI3K)-Akt pathway, mediate inflammatory cytokine production in lipopolysaccharide-stimulated macrophages^16-19. Several of these pathways, including the mitogen-activated protein kinases (MAPK) and nuclear factor-kappa B (NF-?B), have also been studied in response to wear particles and have been shown to contribute to their biological activity^20-22. Although the role of the PI3K-Akt pathway in response to wear particles has not been studied previously, it is involved in many important cell functions, including cell motility, cell survival, and inflammation²³. PI3K is activated in macrophages in response to lipopolysaccharide¹⁸. PI3K activates Akt through a series of coordinated events resulting in phosphorylation at Ser⁴⁷³ and Thr308²³ (Fig. 1). Once activated, Akt has many downstream targets that mediate its regulatory functions^24,25. The purpose of our study was to determine whether titanium particles activate the PI3K-Akt pathway and to determine whether this pathway is important for titanium particle-induced TNF-a production.

Materials and Methods

Materials and Methods | Results | Discussion | References

Titanium Particle Preparation

Commercially pure titanium particles were obtained from Johnson Matthey (number 00681, lot G11G04; Ward Hill, Massachusetts). Ninety percent of the titanium particles were <3.6 µm in diameter, 75% were <2.4 µm, 50% were <1.6 µm, 25% were <1.2 µm, and 10% were <1.1 µm, as measured with a Coulter Counter (Multisizer 3; Beckman Coulter Particle Characterization Laboratory, Miami, Florida). Titanium particles sterilized in 70% ethanol contained substantial amounts of adherent gram-negative-derived endotoxin (36 U of endotoxin per 10⁹ particles) as measured with the high-sensitivity version of the Chromogenic Limulus Amoebocyte Lysate assay (QCL-1000; BioWhittaker, Walkersville, Maryland) supplemented with a ß-glucan blocking reagent (ß-G Blocker; BioWhittaker)^11,26. Virtually endotoxin-free titanium particles were prepared by removal of >99.9% of the adherent endotoxin without detectably altering the size or shape of the particles by alternating incubations in nitric acid and alkali ethanol as previously described¹¹. Titanium particles with adherent endotoxin and so-called endotoxin-free titanium particles were stored (4°C) until use at a concentration of 2 ×10⁷ particles per milliliter in phosphate-buffered saline solution supplemented with penicillin (100 U/mL) and streptomycin (100 µg/mL). For experiments, the particle stock solution was diluted 1250-fold with serum-free culture media and was preincubated (5% CO₂ at 37°C) for one hour. As a control, phosphate-buffered saline solution with antibiotics, but without particles, was equivalently diluted in serum-free media.

Cell Culture

The culture media used throughout was minimum essential media with Earle's salts (Hyclone, Logan, Utah), supplemented with 2 mM L-glutamine (Mediatech, Herndon, Virginia), nonessential amino acids (Mediatech), 100 U/mL penicillin (Mediatech), and 100 µg/mL streptomycin (Mediatech). Unless otherwise specified, all culture media also contained 10% fetal bovine serum (Hyclone). Serum-free culture media contained 0.1% bovine serum albumin (Sigma, St. Louis, Missouri). Calcium and magnesium-free Dulbecco phosphate-buffered saline solution was obtained from Mediatech. All of these reagents were tested for endotoxin with use of the high-sensitivity version of the Chromogenic Limulus Amoebocyte Lysate assay (QCL-1000; BioWhittaker) and were from lots that contained the lowest concentration of endotoxin available.

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Fig. 1: Activation of the PI3K-Akt pathway. See text for details. PI3K = phosphoinositol-3-kinase, PIP2 = phosphatidylinositol,4,5-bisphosphate, PIP3 = phosphatidylinositol-3,4-5-triphosphate, and PDK1 = phosphatidylinositol dependent kinase-1.

The RAW264.7 murine macrophages (ATCC, Manassas, Virginia) were maintained by culture (5% CO₂ at 37°C) in Petri dishes. Prior to the experiments, the RAW264.7 cells were plated (2.4 × 10⁶ cells per 58 cm² dish) in tissue-culture dishes and were cultured for eighteen to twenty hours to allow the cells to adhere to the plates. Cultures were supplemented with the diluted titanium particle suspensions described in the previous paragraph to a final concentration of 3.3 × 10⁶ particles/cm² (approximately eighty particles per cell) or with the control media without particles. Previous studies have shown that this particle concentration produces a robust TNF-a response by RAW264.7 cells²⁷. In selected experiments, RAW264.7 cells were cultured with titanium particles in the presence or absence of specific inhibitors of PI3K or Akt. For this purpose, cultures were supplemented with the indicated concentrations of LY294002 (Calbiochem, La Jolla, California), SH-5 (Calbiochem), or equivalent concentrations of dimethyl sulfoxide (DMSO; Sigma) vehicle during the incubation with titanium particles as well as during a preincubation period prior to the addition of the particles (one hour for LY294002 and two hours for SH-5). In all cases, the final cultures contained 12 mL of serum-containing media (added with the RAW264.7 cells) and 12 mL of serum-free media (added with the titanium particles and the inhibitors).

Assays of TNF-a Production and Cytotoxicity

After culture for the indicated periods of time, the media were harvested and centrifuged to remove cellular debris (twenty-five minutes times 6400 g). Aliquots were stored at —20°C for TNF-a measurement by enzyme-linked immunosorbent assay (ELISA) with use of capture and detection antibodies (AF-410-NA and BAF410; R and D Systems, Minneapolis, Minnesota) and Poly-HRP20-Streptavidin conjugate (RDI-PHRP20-SA2; Research Diagnostics, Concord, Massachusetts) as previously described⁴. Additional aliquots of media were stored for no more than three days at 4°C for cytotoxicity assessment with use of a lactate dehydrogenase (LDH) detection kit (Roche, Mannheim, Germany). The percent cytotoxicity was calculated by comparison with cultures lysed with 1% Triton X-100 as recommended by the manufacturer.

Western Blot Analysis

The RAW264.7 cells were washed twice with ice-cold phosphate-buffered saline solution containing 1 mM sodium orthovanadate and then were lysed in ice-cold buffer containing 1% Triton X-100, 20 mM Tris (pH 7.5), 150 mM NaCl, 1 mM ß-glycerophosphate, 1 mM EDTA, 1 mM EGTA, 2.5 mM sodium pyrophosphate, 1 mM sodium orthovanadate, 1 ng/mL leupeptin, and 1 mM PMSF (phenylmethylsulfonyl fluoride). One protease inhibitor cocktail tablet (Complete Mini; Roche) was added to every 10 mL of lysis buffer. Cell lysates were sonicated four times for five seconds each on ice at a setting of 8 with use of a sonic probe (60 Sonic Dismembrator; Fisher Scientific, Pittsburgh, Pennsylvania). Sonicated lysates were centrifuged (ten minutes × 10,300 g) and stored at —80°C. Total protein was assayed with use of the BCA protein assay kit (Pierce, Rockford, Illinois). Aliquots of cell lysates containing 10 µg of protein were subjected to SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and were electrotransferred to a polyvinylidene difluoride membrane (PVDF; BioRad, Hercules, California). In selected experiments, cell lysates containing 200 µg of protein were immunoprecipitated with use of an anti-Akt antibody that binds to all three Akt isoforms (Upstate, Waltham, Massachusetts). The cell lysates and the immunoprecipitates were subjected to electrophoresis and electrotransferred to a polyvinylidene difluoride membrane as described above. After electrotransfer, polyvinylidene difluoride membranes were incubated for one hour with block buffer (Tris-buffered saline solution, 0.1% Tween-20, and 5% w/v nonfat dry milk), washed with Tris-buffered saline solution with 0.1% Tween-20 (TBS-T) buffer three times for seven minutes each and then probed with a 1:500 dilution of anti-phospho-Ser⁴⁷³ or 1:1000 anti-phospho-Thr³⁰⁸ (Cell Signaling Technology, Beverly, Massachusetts) in phosphate-buffered saline solution containing 5% bovine serum albumin overnight at 4°C. Bound antibodies were detected with HRP (horseradish peroxidase)-linked anti-rabbit IgG (immunoglobulin G) secondary antibody (Cell Signaling Technology) and enhanced chemiluminescence reagents (Amersham Biosciences, Piscataway, New Jersey). The polyvinylidene difluoride membranes were stripped by washing with 0.2 M NaOH for fifteen minutes followed by three washes with TBS-T for seven minutes each. The stripped membranes were incubated for one hour in block buffer and then were reprobed with a 1:1000 dilution of Akt antibody (Cell Signaling Technology) with 5% bovine serum albumin in phosphate-buffered saline solution overnight at 4°C. Bound antibodies were again detected with HRP-linked anti-rabbit IgG secondary antibody and enhanced chemiluminescence reagents.

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Fig. 2: Titanium (Ti) particles with adherent endotoxin increased Akt activation (A and B) and TNF-a production (C) while so-called endotoxin-free titanium particles did not (B and C). Samples used to assess phosphorylation at Thr³⁰⁸ were immunoprecipitated prior to blotting (B). Data from Figures 2, B and C are from the same experiment. The asterisk denotes a significant difference (p < 0.05) compared with groups without titanium particles. The pound sign denotes a significant difference (p < 0.05) compared with the group with endotoxin-free titanium particles at the same time-point. LPS = lipopolysaccharide.

Statistics

All figures are representative of at least three experiments. All Western blots depict representative bands from triplicate cultures. All TNF-a and cytotoxicity data are presented as the mean and the standard error of the mean of triplicate cultures, each assayed in triplicate. Statistical analysis was performed by analysis of variance with use of SuperANOVA software (Abacus Concepts, Berkley, California). Bonferroni-Dunn (control) post hoc tests were used when a single control group was compared with all other groups (Figs. 3, 4, and 5, B). The Fisher protected least significant difference post hoc tests were used when multiple groups were compared with each other (Figs. 2, C and 5, A).

Results

Materials and Methods | Results | Discussion | References

Activation of the PI3K-Akt Pathway by Titanium Particles with Adherent Endotoxin

To assess activation of the PI3K-Akt pathway, we examined Akt phosphorylation at the Ser⁴⁷³ and the Thr³⁰⁸ sites since phosphorylation at these sites is required for Akt activity²³. Titanium particles with adherent endotoxin transiently increased Akt phosphorylation (Fig. 2, A, top two panels). Increased phosphorylation at the Ser⁴⁷³ site is first observed thirty minutes after stimulation, peaked at sixty minutes, and returned to baseline by ninety minutes (Fig. 2, A [top panel] and Fig. 2, B [top panel]). Increased phosphorylation at the Thr³⁰⁸ site was maximal at forty-five minutes after stimulation and returned to baseline at ninety minutes (Fig. 2, A [middle panel]). Akt phosphorylation was also increased by incubation with lipopolysaccharide, which was included as a positive control (Fig. 2, A [top two panels]). Equivalent sample loading was demonstrated by stripping the phospho-Ser⁴⁷³ and phospho-Thr³⁰⁸ blots, then, reprobing for total Akt levels (Fig. 2, A [bottom panel]). In addition, Akt activation in response to endotoxin-free titanium particles was compared with titanium particles with adherent endotoxin. Endotoxin-free particles did not increase phospho-Ser⁴⁷³ Akt activation (Fig. 2, B [top panel]). Equivalent sample loading was again demonstrated by stripping phospho-Ser⁴⁷³ blots and reprobing for total Akt levels (Fig. 2, B [bottom panel]). TNF-a production did not increase after stimulation with endotoxin-free particles (Fig. 2, C). Moreover, Akt phosphorylation in response to titanium particles with adherent endotoxin preceded TNF-a secretion, which was first detectable at sixty minutes (Fig. 2, C).

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Fig. 3: The specific PI3K inhibitor, LY294002, dose-dependently reduced TNF-a production and Akt activation (A) without increasing cytotoxicity (B). Cells were pretreated with or without LY294002 (one hour) followed by stimulation with titanium (Ti) particles with adherent endotoxin for ninety minutes in the continuous presence or absence of LY294002. All groups contained 1% DMSO (dimethyl sulfoxide) as the vehicle control. The pound sign denotes a significant difference (p < 0.05), and the asterisk denotes a significant difference (p < 0.0001) compared with the titanium with endotoxin group without LY294002 (A). No significant difference in cytotoxicity was identified between any two groups (B).

Effect of PI3K-Akt Inhibitors on the Production of TNF-a in Response to Titanium Particles with Adherent Endotoxin

To assess the role of the PI3K-Akt pathway in the production of TNF-a in response to titanium particles with adherent endotoxin, we examined the effects of LY294002, which is a specific inhibitor of PI3K²⁸. For example, LY294002 does not inhibit any of the mitogen-activated protein kinase (MAPK) pathway kinases²⁸, which are known to be stimulated by titanium particles with adherent endotoxin^22,29. LY294002 inhibited TNF-a production in a dose-dependent manner with 70% inhibition at 100 µM (Fig. 3, A). LY294002 also inhibited Akt phosphorylation at the phospho-Ser⁴⁷³ site in a similar dose-dependent manner with a maximal effect at 100 µM (Fig. 3, A). Inhibition at the phospho-Thr³⁰⁸ site displayed slight but consistent inhibition at all LY294002 concentrations. This experiment examined Akt phosphorylation from cells incubated with titanium particles with adherent endotoxin for ninety minutes. The results (Fig. 3, A) confirm the findings in Figure 2, A and B, that the increase in Akt phosphorylation induced by particles occurs prior to this time-point. Cytotoxicity was not increased with the LY294002 concentrations used in these experiments (Fig. 3, B).

We also studied the effects of SH-5, which is a specific inhibitor of Akt activation³⁰. Although this inhibitor has not been tested as rigorously as LY294002, Kozikowski et al. showed that it is a highly specific inhibitor of Akt activity³⁰. Like LY294002, SH-5 does not block the activity of other MAPK pathway kinases, like ERK1/2 or p38, which are activated in response to titanium particles with adherent endotoxin. SH-5 inhibited TNF-a production in a dose-dependent manner with 83% inhibition at 75 µM (Fig. 4, A). Akt phosphorylation at the phospho-Ser⁴⁷³ site was also inhibited in a similar dose-dependent manner by SH-5 with a maximal effect at 75 µM (Fig. 4, A). There was near total inhibition of Akt phosphorylation at the phospho-Thr³⁰⁸ site. Cytotoxicity was not increased by the concentrations of SH-5 used in these experiments (Fig. 4, B).

Effect of LY294002 on TNF-a Production in Response to Endotoxin-Free Titanium Particles

As shown in Figure 5, A, high concentrations (16.5 × 10⁶ particles/cm²) of so-called endotoxin-free titanium particles induce TNF-a production. However, the production of TNF-a occurs much more slowly and to a lesser extent than the response to a fivefold lower concentration of titanium particles with adherent endotoxin. To examine the role of the PI3K-Akt pathway in response to endotoxin-free titanium particles, a high concentration of endotoxin-free particles was used to stimulate RAW264.7 cells. Although there was no detectable increase in Akt phosphorylation with endotoxin-free particles at any of the time-points measured between zero and 180 minutes (data not shown), LY294002 (100 µM) completely inhibited TNF-a production induced by these particles (Fig. 5, A). LY294002 increased cytotoxicity by a significant (p < 0.0001) but small amount that does not account for its inhibition of TNF-a production (Fig. 5, B).

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Fig. 4: The specific Akt inhibitor, SH-5, dose-dependently reduced TNF-a production and Akt activation (A) without increasing cytotoxicity (B). Cells were pretreated with or without SH-5 (two hours) followed by stimulation with titanium (Ti) particles with adherent endotoxin for ninety minutes in the continuous presence or absence of SH-5. All groups contained 0.76% DMSO (dimethyl sulfoxide) as the vehicle control. The asterisk denotes a significant difference (p < 0.0001) compared with the titanium with endotoxin group without SH-5 (A). No significant difference in cytotoxicity between any two groups was seen (B).

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Fig. 5: LY294002 inhibits TNF-a production induced by endotoxin-free titanium (Ti) particles (A) and causes a small increase in cytotoxicity that does not account for the inhibition of TNF-a production (B). Cells were pretreated with or without LY294002 (one hour) followed by stimulation with endotoxin-free titanium particles (16.5 x 10⁶/cm²) for three hours or titanium particles with adherent endotoxin (3.3 x 10⁶/cm²) for one hour in the continuous presence or absence of LY294002. All groups contained 1% DMSO (dimethyl sulfoxide) as the vehicle control. The pound sign denotes a significant difference (p < 0.0001) compared with the titanium with endotoxin group, and the asterisk denotes a significant difference (p < 0.0001) compared with the endotoxin-free titanium group without LY294002.

Discussion

Materials and Methods | Results | Discussion | References

Titanium particles with adherent endotoxin activate the PI3K-Akt signaling pathway in vitro to produce TNF-a. Akt is activated prior to the onset of TNF-a production, and inhibitors of PI3K and Akt activity block titanium particle-induced TNF-a. Furthermore, higher concentrations of endotoxin-free particles over a longer incubation period produce a small but significant (p < 0.0001) increase in TNF-a that is also blocked by inhibition of PI3K. Although no Akt activation was detectable by Western blot in response to endotoxin-free particles at any time-point that we examined, it is possible that we missed the time-point within the three-hour period that Akt activation increased. Alternatively, the magnitude of Akt activation may be low and thus difficult to detect by Western blot. Also, basal levels of Akt activity can exert potent downstream effects and may be required to generate a response to an external stimulus³¹. Nonetheless, our results show that the PI3K-Akt signaling pathway contributes to the induction of TNF-a by wear particles, irrespective of the presence or absence of adherent endotoxin.

We measured TNF-a as a marker for inflammation rather than other cytokines because of its well-documented role in wear particle-induced bone loss^20,32-34. Moreover, TNF-a is the first proinflammatory cytokine produced after macrophage stimulation with wear particles^29,35. TNF-a then stimulates macrophages to increase production of other proinflammatory cytokines³⁶. Therefore, measuring TNF-a as it begins to increase provides a more accurate assessment of the direct particle effect on cytokine production than measuring the secondary effects of TNF-a as it stimulates other cytokines.

Although polyethylene particles are the most prevalent type of particles in periprosthetic tissue^1,37, we used titanium particles in our experiments for three reasons. First, titanium particles are present in periprosthetic tissues from patients undergoing revision total joint arthroplasty³⁷. Second, titanium and polyethylene particles induce osteolysis by similar mechanisms³⁸. Third, polyethylene is difficult to use in cell culture because the particles tend to float in the culture media and, therefore, do not reach the macrophages adherent to the bottom of the culture plate. Thus, titanium particles serve as an appropriate stimulus in a cell culture model.

Although gram-positive bacteria have been found in the biofilm surrounding implants removed for aseptic loosening, we studied the effects of gram-negative endotoxins rather than lipoteichoic acid or peptidoglycan. As previously mentioned, gram-positive and gram-negative endotoxins have similar biological activity^8,9. However, the assays available to measure lipoteichoic acid and peptidoglycan are not sensitive and specific enough to quantify the amount of these bioactive molecules on particles or, more importantly, to document the removal of these molecules from particles²⁶. Assays to measure gram-negative endotoxin are very sensitive and are more accurate for assessing the particle effect compared with the endotoxin effect on TNF-a production.

Other pathways, including nuclear factor-kappa B (NF-?B) and MAPKs, are activated in macrophages stimulated with lipopolysaccharide^16,19. Adherent endotoxin on titanium particles increases activation of NF-?B and all three major mitogen-activated protein kinases (ERK1/2, JNK, and p38)^22,29. Of these, activation of NF-?B and ERK1/2 is required for TNF-a production and inflammation in response to wear particles^20-22. The interaction between NF-?B, ERK1/2, and the PI3K-Akt pathways is likely complex. Figure 6, A summarizes these mechanisms of titanium particle-induced TNF-a production in macrophages. In addition, on the basis of what is known about macrophage stimulation with lipopolysaccharide^16-19, Figure 6, B outlines a proposed mechanism by which the NF-?B, ERK1/2, and the PI3K-Akt pathways interact to increase TNF-a production. Studies are in progress in our laboratory to clarify the cross-talk between these pathways in response to particles. Nonetheless, the current evidence indicates that local or systemic inhibition of any of these pathways could result in the reduction of osteolysis and aseptic loosening. Furthermore, in vivo studies are needed to clarify the consequences of inhibiting these pathways and to determine whether there is a resultant reduction of particle-induced bone loss.

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Fig. 6: Titanium (Ti) particles activate ERK1/2, NF-?B, and the PI3K-Akt signaling pathways in macrophages to increase production of TNF-a (A). Potential interactions among the ERK1/2, NF?-B, and PI3K-Akt signaling pathways are shown (B). See text for details. TLR = Toll-like receptor, IkB = inhibitor of kappa B, IKK = inhibitor of kappa B kinase, Elk1 = Ets-like transcription factor 1, and Egr-1 = Early growth response 1.

LY294002, the inhibitor that was used in our experiments, inhibits multiple isoforms of PI3K^39,40. This agent therefore is potentially lethal in vivo, since mice lacking the genes for either PI3Ka or PI3Kß isoforms die during embryologic development^41,42. In contrast, mice lacking the PI3K? gene show no adverse phenotype and are protected from joint destruction related to rheumatoid arthritis⁴³. An oral drug that selectively blocks PI3K? and the resultant Akt phosphorylation, and thereby protects mice from joint inflammation and destruction in models of rheumatoid arthritis, has recently been developed⁴³. Since our data show that the PI3K-Akt pathway mediates the biological response to wear particles in vitro, this new drug provides an exciting avenue for further research into pharmacological inhibition of aseptic loosening. ?

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Topics

endotoxins ; 1-phosphatidylinositol 3-kinase ; titanium ; signal pathway ; cytotoxicity ; tumor necrosis ; proto-oncogene proteins c-akt ; tumor necrosis factor-alpha

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Matthew V. Smith, Michael J. Lee, Andrew S. Islam, Jacqueline L. Rohrer, Victor M. Goldberg, Michelle A. Beidelschies, Edward M. Greenfield; Inhibition of the PI3K-Akt Signaling Pathway Reduces Tumor Necrosis Factor-α Production in Response to Titanium Particles in Vitro. The Journal of Bone & Joint Surgery. 2007 May;89(5):1019-1027.

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