Titanium inhibits both interferon-gamma and interleukin-6 signaling in human osteoclast precursor cells, whereas polymethylmethacrylate bone cement inhibits only the latter. Wear particle inhibition of interleukin-6 specifically involves the activation of p38 mitogen-activated protein kinase and is accompanied by substantial induction of SOCS3, an inhibitor of interleukin-6 signaling. In contrast, titanium inhibition of interferon-gamma signaling is not dependent on mitogen-activated protein kinase activation and is accompanied by only modest induction of the interferon-gamma inhibitor SOCS1.

The ␥/-chain family of proteins mediate cell activation for multiple immunoglobulin receptors. However, the recognition that these receptors may have distinct biologic functions suggests that additional signaling elements may contribute to functional diversity. We hypothesized that the cytoplasmic domain (CY) of the ligand binding ␣-chain alters the biological properties of the receptor complex. Using macrophage Fc␥RIa as a model system, we created stable transfectants expressing a full-length or a CY deletion mutant of human Fc␥RIa. Both receptors functionally associate with the endogenous murine ␥-chain. However, we have established that the CY of Fc␥RIa directly contributes to the functional properties of the receptor complex. Deletion of the Fc␥RIa CY leads to slower kinetics of receptorspecific phagocytosis and endocytosis as well as lower total phagocytosis despite identical levels of receptor expression. Deletion of the CY also converts the phenotype of calcium independent Fc␥RIa-specific phagocytosis to a calcium-dependent phenotype. Finally, deletion of the CY abrogates Fc␥RIa-specific secretion of interleukin-6 but does not affect production of interleukin-1␤. These results demonstrate a functional role for the CY of Fc␥RIa and provide a general model for understanding how multiple receptors that utilize the ␥-chain can generate diversity in function.Fc␥ receptors play a central role in the handling of immune complexes, regulation of inflammatory responses, antibody secretion, and T cell activity (1-4). Common to each of these functions is the initiation of tyrosine phosphorylation following receptor cross-linking (5) and the involvement of the ␥/ subunits leading to the view that Fc receptors subserve redundant signaling functions. However, recent evidence suggests that these receptors are not redundant. For example, Fc␥RIIIa appears necessary for initiating the Arthus inflammatory reaction (6, 7), while Fc␥RIa and Fc␣RI can down-regulate inflammatory responses by initiating the secretion of IL 1 -10 and IL-1ra, respectively (4, 8). The basis for these differences are unknown.Fc␥RI is expressed on the cell surface in association with the ␥-chain (9, 10). This association is not a prerequisite for transient receptor expression but is necessary for stable expression (11, 12). The ␥-chain cytoplasmic domain contains an immunoreceptor tyrosine activation motif (ITAM) and current data suggest that the ␥-chain cytoplasmic domain is both necessary and sufficient for Fc␥RIa induced functions (13-15). Biochemical studies have shown that cross-linking of the Fc␥RIa⅐␥-chain complex results in activation of a Src family kinase(s) and the tyrosine kinase p72Syk (2,5). Activation of these kinases results in tyrosine phosphorylation of the ␥-chain and the initiation of a signaling cascade that can culminate in the induction of degranulation, phagocytosis, an oxidative burst, ADCC activity and the induction of gene transcription. The association between Fc␥RIa and ␥-chain may also be important in the formation of a h...

The biologic response to particulate load after arthroplasty has not been fully characterized but is believed mediated by proinflammatory cytokines released from mononuclear cells in the periprosthetic region. To investigate the contribution of lymphocytes to expression of proinflammatory genes induced by metal particles, we compared gene expression of mononuclear cells in response to metal and polymethylmethacrylate particles using cDNA microarray profiling. Peripheral blood mononuclear cells and monocytes were stimulated with polymethylmethacrylate and titanium particles of clinically relevant sizes. Polymethylmethacrylate elicited a six- to 12-fold increase in gene expression of tumor necrosis factor alpha, interleukin 1alpha, interleukin 1beta, interleukin 6, and interleukin 8 in purified monocytes and unfractionated peripheral blood mononuclear cells. Although the effect of titanium on stimulation of purified monocytes was modest, stimulation of lymphocyte-containing peripheral blood mononuclear cells by titanium particles resulted in monocyte-derived proinflammatory cytokine expression. In contrast to polymethylmethacrylate, titanium particles stimulated increased expression of T lymphocyte-derived cytokines, including interleukin 2, interferon gamma, interleukin 9, and interleukin 22, in peripheral blood mononuclear cell cultures. The induction of T cell activation by titanium particles suggests lymphocytes may contribute to the inflammation that mediates osteolysis in patients with metallic particulate debris after total joint replacement.

The ability of prosthetic wear debris to induce pro-inflammatory responses in macrophages is widely appreciated, but little is known about the molecular mechanisms involved in particle recognition. Specifically, the nature of the cell surface receptors that interact with wear debris is poorly understood. Elucidating the identities of these receptors and how they interact with different types of wear debris are critical to understanding how wear debris initiates periprosthetic osteolysis. We examined the involvement of opsonization, complement receptor 3 (CR3), and scavenger receptor A (SRA), in responses to polymethylmethacrylate (PMMA) and titanium wear particles. Serum dependence of pro-inflammatory responses to PMMA and titanium was tested, and serum proteins that adhered to these two types of particles were identified. Several serum proteins, including known opsonins such as C3bi and fibronectin, adhered to PMMA but not titanium, and serum was required for pro-inflammatory signaling induced by PMMA, but not by titanium. Phagocytosis of PMMA and titanium by macrophages was demonstrated by flow cytometry. Blocking CR3 specifically inhibited phagocytosis of PMMA by macrophages, whereas blocking SRA specifically inhibited titanium uptake. Direct involvement of CR3 and SRA in cell-particle interaction was assessed by expression of these receptors in nonphagocytic HEK293 cells. CR3 specifically induced cell binding to PMMA particles and adhesion to PMMA-coated plates, while SRA specifically induced binding to titanium particles and adhesion to titanium-coated plates. Taken together, these results suggest involvement of opsonization, complement, and integrin receptors, including CR3 and fibronectin receptors, in PMMA action, and an involvement of scavenger receptors in responses to titanium. ß

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