Kenneth A. Roebuck

Regulation of intercellular adhesion molecule‐1 (CD54) gene expression

Intercellular adhesion molecule‐1 (ICAM‐1, CD54) is an inducible cell adhesion glycoprotein of the immunoglobulin supergene family expressed on the surface of a wide variety of cell types. ICAM‐1 interactions with the β2 integrins CD11a/CD18 (LFA‐1) and CD11b/CD18 (MAC‐1) on the surface of leukocytes are important for their transendothelial migration to sites of inflammation and their function as costimulatory molecules for T cell activation. ICAM‐1 is constitutively expressed on the cell surface and is up‐regulated in response to a variety of inflammatory mediators, including proinflammatory cytokines, hormones, cellular stresses, and virus infection. These stimuli increase ICAM‐1 expression primarily through activation of ICAM‐1 gene transcription. During the past decade much has been learned about the cell type‐ and stimulus‐specific transcription of ICAM‐1. The architecture of the ICAM‐1 promoter is complex, containing a large number of binding sites for inducible transcription factors, the most important of which is nuclear factor‐kappa B (NF‐κB). NF‐κB acts in concert with other transcription factors and co‐activators via specific protein‐protein interactions, which facilitate the assembly of distinct stereo‐specific transcription complexes on the ICAM‐1 promoter. These transcription complexes presumably mediate the induction of ICAM‐1 expression in different cell types and in response to different stimuli. In this review, we summarize our current understanding of ICAM‐1 gene regulation with a particular emphasis on the transcription factors and signal transduction pathways critical for the cell type‐ and stimulus‐specific activation of ICAM‐1 gene transcription. J. Leukoc. Biol. 66: 876–888; 1999.

Osteolysis: basic science.

Since the recognition of aseptic loosening by Charnley in the early 1960s, much information has been gained on the basic science of periprosthetic bone loss. Initially termed cement disease, it now generally is accepted that, in most instances, osteolysis is a manifestation of an adverse cellular response to phagocytosable particulate wear and corrosion debris, possibly facilitated by local hydrodynamic effects. Tissue explant, animal, and cell culture studies have allowed us to compile an appreciation of the complexity of cellular interactions and chemical mediators involved in osteolysis. Cellular participants have been shown to include the macrophage, osteoblast, fibroblast, and osteoclast. The plethora of chemical mediators that are responsible for the cellular responses and effects on bone include prostaglandin E2, tumor necrosis factor-alpha, interleukin-1, and interleukin 6. However, an increasing number of other proinflammatory and antiinflammatory cytokines, prostenoids, and enzymes have been shown to play important roles in this process. The ultimate goal of basic research is to develop novel strategies for evaluation and treatment of patients with osteolysis. Although initial animal studies are promising for possible pharmacologic treatment and prevention of osteolysis, well-controlled human trials are required before agents such as bisphosphonates can be recommended for general clinical use.

Stimulus-specific regulation of chemokine expression involves differential activation of the redox-responsive transcription factors AP-1 and NF-kappaB.

The promoters of the IL‐8, MCP‐1, and RANTES genes contain binding sites for the redox‐responsive transcription factors AP‐1 and NF‐κB, which have been shown to be important for their expression. In this overview, we present evidence from our laboratories that the stimulus‐specific regulation of these chemokines by the reactive oxidant H2O2, the proinflammatory cytokine TNF‐α, and respiratory syncytial virus (RSV) is mediated in a cell type‐specific manner involving different patterns of AP‐1 and NF‐κB binding activity. Our results demonstrate that H2O2 induction of IL‐8 gene expression is linked with the selective binding of AP‐1 to the IL‐8 promoter, whereas TNF‐α and RSV induction of IL‐8 correlates with the activation of NF‐κB binding. We propose that the differential activation and binding of inducible transcription factors to the promoter regions of chemokine genes provides a critical regulatory mechanism by which the CXC and CC chemokines can be selectively expressed in a cell type‐specific and stimulus‐specific manner. Such a regulatory mechanism of differential chemokine expression could critically influence the site‐ specific recruitment of distinct subsets of leukocytes to sites of inflammation and injury. J. Leukoc. Biol. 65: 291–298; 1999.

The Effects of Particulate Wear Debris, Cytokines, and Growth Factors on the Functions of MG-63 Osteoblasts

Background: Particle-challenged cells release cytokines, chemokines, and eicosanoids, which contribute to periprosthetic osteolysis. The particle-induced activation of macrophages and monocytes has been extensively studied, but only limited information is available on the response of osteoblasts to particulate wear debris. This study examines the effects of particulate wear debris, proinflammatory cytokines, and growth factors on osteoblast functions. Methods: MG-63 osteoblasts were treated with metal particles (titanium, titanium alloy, and chromium orthophosphate) or polymeric particles (polyethylene and polystyrene) of phagocytosable sizes or were treated with exogenous cytokines and growth factors. The kinetics of particle phagocytosis and the number of engulfed particles were assessed with use of fluoresceinated particles. Cell proliferation was determined according to [3H]-thymidine incorporation, and cell viability was determined by either fluorescein diacetate uptake or trypan blue exclusion. Expressions of osteoblast-specific genes were quantified with Northern blot hybridization, and the secretions of osteoblast-specific proteins and cytokines were analyzed by enzyme-linked immunosorbent assays. Results: MG-63 osteoblasts phagocytosed particles and became saturated after twenty-four hours. A maximum of forty to sixty particles per cell were phagocytosed. Each type of particle significantly suppressed procollagen a1[I] gene expression (p < 0.05), whereas other osteoblast-specific genes (osteonectin, osteocalcin, and alkaline phosphatase) did not show significant changes. Particle-stimulated osteoblasts released interleukin-6 (p < 0.05) and a smaller amount of transforming growth factor-b1. Particles reduced cell proliferation in a dose-dependent manner without affecting cell viability (p < 0.05). Exogenous tumor necrosis factor-a also enhanced the release of interleukin-6 (p < 0.01) and transforming growth factor-b1 (p < 0.05), whereas the secretion of transforming growth factor-b1 was increased by insulin-like growth factor-I and prostaglandin E2 as well. Insulin-like growth factor-I and transforming growth factor-b1 significantly increased procollagen a1[I] gene expression in osteoblasts (p < 0.05), while tumor necrosis factor-a and prostaglandin E2 significantly suppressed procollagen a1[I] gene expression (p < 0.01). In contrast, neither exogenous nor endogenous interleukin-6 had any effect on other cytokine secretion, on proliferation, or on procollagen a1[I] gene expression. The transcription inhibitor actinomycin D reduced both procollagen a1[I] transcription and interleukin-6 production. Inhibitors of protein synthesis (cyclohexamide) and intracellular protein transport (brefeldin A and monensin) blocked the release of interleukin-6, but none of these compounds influenced the suppressive effect of titanium on procollagen a1[I] gene expression. Conclusions: MG-63 osteoblasts phagocytose particulate wear debris, and this process induces interleukin-6 production and suppresses type-I collagen synthesis. Osteoblast-derived interleukin-6 may induce osteoclast differentiation and/or activation, but the resorbed bone cannot be replaced by new bone because of diminished osteoblast function (reduced type-I collagen synthesis). Exogenous cytokines (tumor necrosis factor-a and interleukin-1b), growth factors (insulin-like growth factor-I and transforming growth factor-b1), and prostaglandin E2 can modify particulate-induced alterations of osteoblast functions. Clinical Relevance: Altered osteoblast functions probably contribute to the progression of periprosthetic osteolysis. Suppressed osteoblast functions, however, could be compensated for by certain growth factors, such as insulin-like growth factor-I or transforming growth factor-b1. These growth factors, if delivered locally, may have therapeutic potential to prevent or reverse periprosthetic osteolysis.

The Basic Science of Periprosthetic Osteolysis

Despite improvements in the techniques, materials, and fixation of total joint replacements, wear and its sequelae continue to be the main factors limiting the longevity and clinical success of arthroplasty. Since Charnley first recognized aseptic loosening in the early 1960s, a tremendous amount of information has been gained on the basic science of osteolysis. Tissue explant, animal, and cell culture studies have allowed development of an appreciation of the complexity of cellular interactions and chemical mediators involved in these processes. Cellular participants have been shown to include the macrophage, osteoblast, fibroblast, and osteoclast. The plethora of chemical mediators that are responsible for the cellular interactions and effects on bone primarily include PGE2, TNF-alpha, IL-1, and IL-6. Recent and ongoing work in the field of signaling pathways will continue to advance our understanding of the mechanisms of periprosthetic bone loss. Although initial animal studies are promising for the development of possible pharmacologic agents for the treatment and prevention of osteolysis, well controlled human trials are required.

Differential regulation of interleukin-8 and intercellular adhesion molecule-1 by H2O2 and tumor necrosis factor-alpha in endothelial and epithelial cells.

The reactive oxygen intermediate H2O2 can function as a signaling molecule to activate gene expression. In this study, we demonstrate that oxidant stress induced by tumor necrosis factor α (TNFα) or H2O2 differentially regulates intercellular adhesion molecule-1 (ICAM-1) and interleukin-8 (IL-8) gene expression in endothelial and epithelial cells. Northern blot analysis revealed that TNFα induced both ICAM-1 and IL-8 expression in either the A549 lung epithelial cell line or the human microvessel endothelial cell line (HMEC-1). In contrast, H2O2 selectively induced only ICAM-1 in HMEC-1 and only IL-8 in A549. This cell type-specific pattern of IL-8 expression was also observed in several other endothelial and epithelial cells. TNFα induced greater IL-8 gene expression as compared with H2O2, but the kinetics of induction were similar. The induction of epithelial IL-8 message was accompanied by a corresponding increase in functional IL-8 protein secretion as determined by a neutrophil motility assay. The increased neutrophil motility stimulated by conditioned media from H2O2- or TNFα-exposed A549 cells was completely inhibited by an anti-IL-8 antibody. TNFα and H2O2 also induced a differential pattern of CC chemokine expression in A549. While TNFα induced both RANTES and MCP-1, H2O2 induced only MCP-1. These data suggest that epithelial cells under oxidant stress contribute to the inflammatory cytokine network by selective production of IL-8, MCP-1, and RANTES, which may critically influence the site-specific recruitment of leukocyte subsets.

Particulate Wear Debris Activates Protein Tyrosine Kinases and Nuclear Factor κB, Which Down‐Regulates Type I Collagen Synthesis in Human Osteoblasts

Particulate wear debris generated mechanically from prosthetic materials is phagocytosed by a variety of cell types within the periprosthetic space including osteoblasts, which cells with an altered function may contribute to periprosthetic osteolysis. Exposure of osteoblast‐like osteosarcoma cells or bone marrow‐derived primary osteoblasts to either metallic or polymeric particles of phagocytosable sizes resulted in a marked decrease in the steady‐state messenger RNA (mRNA) levels of procollagen α1[I] and procollagen α1[III]. In contrast, no significant effect was observed for the osteoblast‐specific genes, such as osteonectin and osteocalcin (OC). In kinetic studies, particles once phagocytosed, maintained a significant suppressive effect on collagen gene expression and type I collagen synthesis for up to five passages. Large particles of a size that cannot be phagocytosed also down‐regulated collagen gene expression suggesting that an initial contact between cells and particles can generate gene responsive signals independently of the phagocytosis process. Concerning such signaling, titanium particles rapidly increased protein tyrosine phosphorylation and nuclear transcription factor κB (NF‐κB) binding activity before the phagocytosis of particles. Protein tyrosine kinase (PTK) inhibitors such as genistein and the NF‐κB inhibitor pyrrolidine dithiocarbamate (PDTC) significantly reduced the suppressive effect of titanium on collagen gene expression suggesting particles suppress collagen gene expression through the NF‐κB signaling pathway. These results provide a mechanism by which particulate wear debris can antagonize the transcription of the procollagen α1[I] gene in osteoblasts, which may contribute to reduced bone formation and progressive periprosthetic osteolysis.

Bacterial vaginosis-associated microflora isolated from the female genital tract activates HIV-1 expression.

Alteration of cervicovaginal microbial flora can lead to vaginosis, which is associated with an increased risk of HIV-1 transmission. We recently characterized a soluble HIV-inducing factor (HIF) from the cervicovaginal lavage (CVL) samples of women. The goals of this study were to determine the effect of cervicovaginal microflora on HIV-1 expression and to elucidate the relationship between HIF activity and microflora. Physiologically relevant microorganisms, Mycoplasma, diphtheroid-like bacteria, Gardnerella vaginalis, Streptococcus agalactiae, and Streptococcus constellatus, cultured from the CVL of a representative woman with a clinical condition of bacterial vaginosis and possessing HIF activity, induced HIV-1 expression. The magnitude of virus induction varied widely with the greatest stimulation induced by diphtheroid-like bacteria and Mycoplasma. The transcriptional induction by Mycoplasma was mediated by activation of the KB enhancer, an activation mechanism shared with HIF. Also as with HIF, Mycoplasma induced AP-1 dependent transcription. Polymerase chain reaction (PCR)-based speciation showed that the isolate was M. hominis. Our data indicate that bacterial vaginosis-associated microflora can enhance HIV-1 transcription and replication and identify M. hominis as a potential source for HIF activity. The virus-enhancing activities associated with the microflora and HIF may increase genital tract viral load, potentially contributing to HIV transmission.

Titanium particles induce the immediate early stress responsive chemokines IL-8 and MCP-1 in osteoblasts.

Exposure of human osteoblasts to ultrafine titanium (Ti) particles has been shown to alter osteoblast gene expression. We previously reported that Ti particles can increase IL‐6 release and suppress the gene expression of procollagens α1[I] and α1[III] in human osteoblasts. In this study, we now demonstrate that Ti particles can rapidly induce the chemotactic cytokines interleukin‐8 (IL‐8) and monocyte chemoattractant protein‐1 (MCP‐1), two immediate early stress responsive chemokines important for the activation and chemotaxis of neutrophils and macrophages, respectively. In MG‐63 osteosarcoma cells and bone marrow derived primary osteoblasts Ti particles selectively increased the steady state levels of IL‐8 and MCP‐1 mRNA in a time and concentration dependent manner. The increased chemokine mRNA correlated with increased secretion of IL‐8 and MCP‐1 protein. Actinomycin D, a potent RNA polymerase II inhibitor, blocked the Ti particle induction of IL‐8 and MCP‐1 mRNA expression, whereas cycloheximide, which inhibits protein synthesis, failed to inhibit chemokine gene expression suggesting Ti particles directly target activation of chemokine gene transcription. Consistent with a transcriptional mechanism not involving new protein synthesis, we demonstrate that Ti particles induce the binding of the p65 and p50 subunits of the latent transcription factor NF‐κB to the IL‐8 gene promoter. Taken together, these data demonstrate that Ti particles can activate transcription of the stress responsive chemokine genes IL‐8 and MCP‐1 in human osteoblasts.

Shedding of the Interleukin-6 (IL-6) Receptor (gp80) Determines the Ability of IL-6 to Induce gp130 Phosphorylation in Human Osteoblasts

Human osteoblasts produce interleukin-6 (IL-6) and respond to IL-6 in the presence of soluble IL-6 receptor (sIL-6R), but the cell surface expression of IL-6R and the mechanism of sIL-6R production are largely unknown. Three different human osteoblast-like cell lines (MG-63, HOS, and SaOS-2) and bone marrow-derived primary human osteoblasts expressed both IL-6R and gp130 as determined by flow cytometry and immunoprecipitation. However, the membrane-bound IL-6R was nonfunctional, as significant tyrosine phosphorylation of gp130 did not occur in the presence of IL-6. Phorbol myristate acetate induced a dramatic increase of both IL-6R shedding (i.e. the production of sIL-6R) and IL-6 release in osteoblast cultures, but the cell surface expression of gp130 remained unchanged. IL-6 complexed with sIL-6R, either exogenously introduced or derived from the nonfunctional cell surface form by shedding, induced rapid tyrosine phosphorylation of gp130. This effect was inhibited by neutralizing antibodies to either sIL-6R or gp130, indicating that the gp130 activation was induced by IL-6/sIL-6R/gp130 interaction. Protein kinase C inhibitors blocked phorbol myristate acetate-induced and spontaneous shedding of IL-6R resulting in the absence of sIL-6R in the culture medium, which in turn also prevented the activation of gp130. In conclusion, human osteoblasts express cell surface IL-6R, which is unable to transmit IL-6-induced signals until it is shed into its soluble form. This unique mechanism provides the flexibility for osteoblasts to control their own responsiveness to IL-6 via the activation of an IL-6R sheddase, resulting in an immediate production of functionally active osteoblast-derived sIL-6R.