title: Signaling pathways for tumor necrosis factor-alpha and interleukin-6 expression in human macrophages exposed to titanium-alloy particulate debris in vitro.
abstract: 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-alpha (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-kappaB) 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-alpha 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-alpha or IL-6. Thus, phagocytosis of particles was not necessary for induction of the release of TNF-alpha or IL-6 in the cultured macrophages. Ligation of the macrophage CD11b/CD18 receptors by integrin-specific antibodies also increased the release of TNF-alpha 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-alpha and IL-6. Blocking RNA synthesis with actinomycin D or preventing protein synthesis with cycloheximide abolished or decreased particle-induced release of TNF-alpha and IL-6 from the macrophages. Macrophage release of TNF-alpha 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-kappaB 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-kappaB and NF-IL-6.
  
  
  CONCLUSIONS
  These data suggest that particle induced macrophage release of TNF-alpha and IL-6 does not require phagocytosis but is dependent on tyrosine and serine/threonine kinase activity culminating in activation of
authors.affiliation: Orthopaedic Research Laboratory, Stanford University Medical Center, California 94305-5341, USA.
authors.forename: Y
authors.initials: Y
authors.lastname: Nakashima
authors.forename: D H
authors.initials: DH
authors.lastname: Sun
authors.forename: M C
authors.initials: MC
authors.lastname: Trindade
authors.forename: W J
authors.initials: WJ
authors.lastname: Maloney
authors.forename: S B
authors.initials: SB
authors.lastname: Goodman
authors.forename: D J
authors.initials: DJ
authors.lastname: Schurman
authors.forename: R L
authors.initials: RL
authors.lastname: Smith
mesh_terms: D000497:Alloys; D015153:Blotting, Western; D022762:CCAAT-Enhancer-Binding Proteins; D004268:DNA-Binding Proteins / Q000378:metabolism; D006622:Hip Prosthesis; D006801:Humans; D066298:In Vitro Techniques; D015850:Interleukin-6 / Q000378:metabolism*; D008262:Macrophage Activation; D008264:Macrophages / Q000378:metabolism*; D016328:NF-kappa B / Q000378:metabolism; D009687:Nuclear Proteins / Q000378:metabolism; D010587:Phagocytosis; D011475:Prosthesis Failure; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D015398:Signal Transduction; D014025:Titanium / Q000494:pharmacology*; D014157:Transcription Factors / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism*
chemical_list: D000497:Alloys; D022762:CCAAT-Enhancer-Binding Proteins; D004268:DNA-Binding Proteins; D015850:Interleukin-6; D016328:NF-kappa B; D009687:Nuclear Proteins; D014157:Transcription Factors; D014409:Tumor Necrosis Factor-alpha; D014025:Titanium; D011505:Protein-Tyrosine Kinases; D017346:Protein Serine-Threonine Kinases