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-a (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-a 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-a or IL-6. Thus, phagocytosis of particles was not necessary for induction of the release of TNF-a or IL-6 in the cultured macrophages. Ligation of the macrophage CD11b/CD18 receptors by integrin-specific antibodies also increased the release of TNF-a 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-a and IL-6. Blocking RNA synthesis with actinomycin D or preventing protein synthesis with cycloheximide abolished or decreased particle-induced release of TNF-a and IL-6 from the macrophages.Macrophage release of TNF-a 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-a 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.