PDX regulates inflammatory cell infiltration via resident macrophage in LPS-induced lung injury.

Inflammatory cell infiltration contributes to the pathogenesis of acute respiratory distress syndrome (ARDS). Protectin DX (PDX), an endogenous lipid mediator, shows anti-inflammatory and proresolution bioactions. In vivo, the mice were intraperitoneally injected with PDX (0.1 µg/mouse) after intratracheal (1 mg/kg) or intraperitoneal (10 mg/kg) LPS administration. Flow cytometry was used to measure inflammatory cell numbers. Clodronate liposomes were used to deplete resident macrophages. RT-PCR, and ELISA was used to measure MIP-2, MCP-1, TNF-α and MMP9 levels. In vitro, sorted neutrophils, resident and recruited macrophages (1 × 106 ) were cultured with 1 µg/mL LPS and/or 100 nmol/L PDX to assess the chemokine receptor expression. PDX attenuated LPS-induced lung injury via inhibiting recruited macrophage and neutrophil recruitment through repressing resident macrophage MCP-1, MIP-2 expression and release, respectively. Finally, PDX inhibition of neutrophil infiltration and transmembrane was associated with TNF-α/MIP-2/MMP9 signalling pathway. These data suggest that PDX attenuates LPS-stimulated lung injury via reduction of the inflammatory cell recruitment mediated via resident macrophages.

Porphyromonas gingivalis lipopolysaccharide induces over production of CC chemokine ligand 2 via toll-like receptor-4 in oral lichen planus.

BACKGROUND: We recently reported that the CC chemokine ligand 2 (CCL2)-CC receptor 2 (CCR2) axis was involved in the pathogenesis of oral lichen planus (OLP). However, the exact mechanism for the high expression of CCL2 in OLP specimens is not clear. Therefore, this study was designed to investigate the potential role of the toll-like receptor 4 (TLR-4) pathway in overproduction of CCL2 in OLP lesions. METHODS: Immunohistochemical staining and real-time RT-PCR were used to detect TLR-4, CCL2, and CCR2 expression in OLP lesions. Then, gingival epithelial cells from OLP lesions were established and treated with Porphyromonas gingivalis (P. gingivalis) lipopolysaccharide (LPS). CCL2 expression in epithelial cells was determined by Western blotting and real-time RT-PCR. In some experiments, TAK-242, a specific inhibitor of TLR-4, was used to block the TLR-4 pathway before cells were stimulated with LPS. RESULTS: We found that TLR-4 was significantly increased in the epithelium of OLP specimens, compared with controls. Moreover, LPS can induce the over production of CCL2 in epithelial cells of OLP, in vitro. TAK-242 effectively eliminated the increase in CCL2 expression induced by LPS by blocking the TLR-4/NF-κB pathway. In addition, we again confirmed that expression of CCL2 and CCR2 was increased in OLP specimens. CONCLUSION: Increased TLR-4 expression contributes to the upregulated expression of CCL2 in the epithelium of OLP lesions, which suggests that oral bacteria participate in the pathogenesis of OLP via the TLR-4 pathway.

Calprotectin Induces IL-6 and MCP-1 Production via Toll-Like Receptor 4 Signaling in Human Gingival Fibroblasts.

Calprotectin, a heterodimer of S100A8 and S100A9 molecules, is associated with inflammatory diseases such as inflammatory bowel disease. We have reported that calprotectin levels in gingival crevicular fluids of periodontitis patients are significantly higher than in healthy subjects. However, the functions of calprotectin in pathophysiology of periodontitis are still unknown. The aim of this study is to investigate the effects of calprotectin on the productivity of inflammatory cytokines in human gingival fibroblasts (HGFs). The HGFs cell line CRL-2014® (ATCC) were cultured, and total RNAs were collected to examine the expression of TLR2/4 and RAGE mRNA using RT-PCR. After the cells were treated with S100A8, S100A9, and calprotectin, supernatants were collected and the levels of IL-6 and MCP-1 were measured using ELISA methods. To examine the intracellular signals involved in calprotectin-induced cytokine production, several chemical inhibitors were used. Furthermore, after the siRNA-mediated TLR4 down-regulated cells were treated with S100A8, S100A9, and calprotectin, the levels of IL-6 and MCP-1 were also measured. HGFs showed greater expression of TLR4 mRNA, but not TLR2 and RAGE mRNA compared with human oral epithelial cells. Calprotectin increased significantly the production of MCP-1 and IL-6 in HGFs, and the cytokine productions were significantly suppressed in the cells treated with MAPKs, NF-κB, and TLR4 inhibitors. Furthermore, calprotectin-mediated MCP-1 and IL-6 production were significantly suppressed in TLR4 down-regulated cells. Taken together, calprotectin induces IL-6 and MCP-1 production in HGFs via TLR4 signaling that involves MAPK and NF-κB, resulting in the progression of periodontitis. J. Cell. Physiol. 232: 1862-1871, 2017. © 2016 Wiley Periodicals, Inc.

Nanoliposomal Buparvaquone Immunomodulates Leishmania infantum-Infected Macrophages and Is Highly Effective in a Murine Model.

Visceral leishmaniasis is a fatal parasitic neglected disease affecting 1.5 million people worldwide. Based on a drug repositioning approach, the aim of this work was to investigate the in vitro immunomodulatory potential of buparvaquone (BPQ) and to establish a safe regimen to evaluate the in vivo efficacy of BPQ entrapped by negatively charged nanoliposomes (BPQ-LP) in Leishmania infantum-infected hamsters. Small-angle X-ray scattering, dynamic light scattering, and the ζ-potential were applied in order to study the influence of BPQ on the liposome structure. Our data revealed that BPQ was located in the polar-apolar interface, snorkeling the polar region, and protected against aggregation inside the lipophilic region. The presence of BPQ also decreased the Z-average hydrodynamic diameter and increased the surface charge. Compared to intravenous and intramuscular administration, a subcutaneous route was a more effective route for BPQ-LP; at 0.4 mg/kg, BPQ-LP reduced infection in the spleen and liver by 98 and 96%, respectively. Treatment for 5 days resulted in limited efficacy, but 10 days of treatment resulted in an efficacy similar to that of a 15-day regimen. The nanoliposomal drug was highly effective, with a mean 50% effective dose of 0.25 mg/kg, reducing the parasite load in bone marrow by 80%, as detected using quantitative PCR analysis. In addition, flow cytometry studies showed that BPQ upregulated cytokines as tumor necrosis factor, monocyte chemoattractant protein 1, interleukin-10 (IL-10), and IL-6 in Leishmania-infected macrophages, eliminating the parasites via a nitric oxide-independent mechanism. This new formulation proved to be a safe and effective treatment for murine leishmaniasis that could be a useful candidate against visceral leishmaniasis.

Interleukin-18 protects mice from Enterovirus 71 infection.

Previous study has demonstrated that the NLRP3 inflammasome is essential for protecting murine host against Enterovirus 71 (EV71) infection. However, the underlying mechanism remained unknown. Here we discovered that the pleiotropic cytokine interleukin-18 (IL-18), an NLRP3 inflammasome-dependent effector protein, exhibits a protective capability against EV71 challenge. Deficiency of IL-18 in mice exacerbated EV71 infection, which was reflected by increased viral replication, elevated production of interferons (IFN-ß, IFN-γ), proinflammatory cytokines (TNF-α, IL-6) and chemokine CCL2,as well as decreased survival of experimental animals. Conversely, administration of recombinant IL-18 considerably restrained EV71 infection in IL-18 deficient mice. Thus, our results revealed a protective role for IL-18 against EV71 challenge, and indicated a novel therapeutic application for IL-18 in EV71 associated hand, foot, and mouth disease (HFMD).

The effects of P. gingivalis and E. coli LPS on the expression of proinflammatory mediators in human mast cells and their relevance to periodontal disease.

Mast cells (MCs) are tissue-resident immune cells that participate in a variety of allergic and inflammatory conditions, including periodontal disease, through the release of cytokines, chemokines and proteolytic enzymes. Porhyromonas gingivalis (P. g) is widely recognized as a major pathogen in the development and progression of periodontitis. Here we compared the differential effects of lipopolysaccharides (LPS) from P. g and E. coli on the expression and production of tumor necrosis factor (TNF), vascular endothelial growth factor (VEGF) and monocyte chemoattractant protein (MCP-1) by human MCs. Human LAD2 MCs were stimulated with LPS from either P. g or E. coli (1-1000 ng/ml). MCs were also stimulated with SP (2µM) serving as the positive control or media alone as the negative control. After 24 h, the cells and supernatant fluids were collected and analyzed for ß-Hexosaminidase (ß-hex) spectrophotometrically, TNF, VEGF and MCP-1 release by ELISA and real-time polymerase chain reaction (PCR) for mediator gene expression, respectively. To assess the functional role of tolllike receptors (TRL) in mediator release, MCs were pre-incubated with either anti-TLR2 or anti- TLR4 (2 µg/ml) polyclonal antibody for 1 h before stimulation with LPS. When MCs were stimulated with SP (2 µM), there was a statistically significant ß-hex release as well as release of TNF, VEGF and MCP-1. Stimulation of MCs with either type of LPS did not induce degranulation based on the lack of ß-hex release. However, both types of LPS stimulated expression and release of TNF, VEGF and MCP-1. Although, P. g LPS induced significant release of TNF, VEGF and MCP-1, the effect was not concentration-dependent. There was no statistically significant difference between the effects of P. g and E. coli LPS. P. g LPS stimulated TNF through TLR-2 while E. coli utilized TRL-4 instead. In contrast, VEGF release by P. g LPS required both TRL-2 and TRL-4 while E. coli LPS required TLR-4. Release of MCP-1 was independent of TLR-2 or TLR-4. P. g LPS activates human MCs to generate and release TNF, VEGF and MCP-1 through different TLRs than E. coli LPS. MCs may, therefore, be involved in the inflammatory processes responsible for periodontal disease.

Focal adhesion kinase knockdown in carcinoma-associated fibroblasts inhibits oral squamous cell carcinoma metastasis via downregulating MCP-1/CCL2 expression.

Carcinoma-associated fibroblasts (CAFs) have been demonstrated to play an important role in the occurrence and development of oral squamous cell carcinoma (OSCC). The aim of this study is to investigate the influence of CAFs on OSCC cells and to explore the role of focal adhesion kinase (FAK) in this process. The results showed that oral CAFs expressed a higher level of FAK than normal human gingival fibroblasts (HGFs), and the conditioned medium (CM) of CAFs could induce the invasion and migration of SCC-25, one oral squamous carcinoma cell line. However, knockdown of FAK by small interfering RNA (siRNA) resulted in inhibition of CAF-CM induced cell invasion and migration in SCC-25, probably by reducing the production of monocyte chemoattractant protein-1 (MCP-1/CCL2), one of downstream target chemokines. Therefore, our findings indicated that targeting FAK in CAFs might be a promising strategy for the treatment of OSCC in the future.

In vivo toxicity of cationic micelles and liposomes.

This study investigated toxicity of nanocarriers comprised of cationic polymer and lipid components often used in gene and drug delivery, formulated as cationic micelles and liposomes. Rats were injected intravenously with 10, 25 or 100 mg/kg and sacrificed after 24 or 48 h, or 24 h after the last of three intravenous injections of 100 mg/kg every other day. Histological evaluation of liver, lung and spleen, clinical chemistry parameters, and hematology indicated little effect of treatment. DNA strand breaks were increased in the lung and spleen. Further, in the dose response study we found unaltered expression levels of genes in the antioxidant response (HMOX1) and repair of oxidized nucleobases (OGG1), whereas expression levels of cytokines (IL6, CXCL2 and CCL2) were elevated in lung, spleen or liver. The results indicate that assessment of genotoxicity and gene expression add information on toxicity of nanocarriers, which is not obtained by histology and hematology. FROM THE CLINICAL EDITOR: This study investigates the toxicity of cationic micelles and liposomes utilized as nanocarriers in gene and drug delivery, demonstrating its effects on the lungs, spleen and liver.

Macrophage proinflammatory response to the titanium alloy equipment in dental implantation.

Titanium alloy and stainless steel (SS) had been widely used as dental implant materials because of their affinity with epithelial tissue and connective tissue, and good physical, chemical, biological, mechanical properties and processability. We compared the effects of titanium alloy and SS on macrophage cytokine expression as well as their biocompatibility. Mouse macrophage RAW264.7 cells were cultured on titanium alloy and SS surfaces. Cells were counted by scanning electron microscopy. A nitride oxide kit was used to detect released nitric oxide by macrophages on the different materials. An enzyme linked immunosorbent assay was used to detect monocyte chemoattractant protein-1 levels. Scanning electron microscopy revealed fewer macrophages on the surface of titanium alloy (48.2 ± 6.4 x 10(3) cells/cm(2)) than on SS (135 ± 7.3 x 10(3) cells/cm(2)). The nitric oxide content stimulated by titanium alloy was 22.5 mM, which was lower than that stimulated by SS (26.8 mM), but the difference was not statistically significant (P = 0.07). The level of monocyte chemoattractant protein-1 released was significantly higher in the SS group (OD value = 0.128) than in the titanium alloy group (OD value = 0.081) (P = 0.024). The transforming growth factor-b1 mRNA expression levels in macrophages after stimulation by titanium alloy for 12 and 36 h were significantly higher than that after stimulation by SS (P = 0.31 and 0.25, respectively). Macrophages participate in the inflammatory response by regulating cytokines such as nitric oxide, monocyte chemoattractant protein-1, and transforming growth factor-b1. There were fewer macrophages and lower inflammation on the titanium alloy surface than on the SS surface. Titanium alloy materials exhibited better biological compatibility than did SS.

Proteolytic activity of Porphyromonas gingivalis attenuates MCP-1 mRNA expression in LPS-stimulated THP-1 cells.

Bacteria can modulate cytokine production of host cells. In this study, we examined effects of Porphyromonas gingivalis, an important periodontal pathogen, on the cytokine production in lipopolysaccharide (LPS)-stimulated THP-1 macrophagic cells. A wide range of doses of P. gingivalis increased the tumor necrosis factor-α (TNF-α) production. However, monocyte chemoattractant protein-1 (MCP-1) production was substantially suppressed by high doses of P. gingivalis and this effect was demonstrated at the mRNA level. Challenges with a congenic protease mutant strain did not significantly attenuate the MCP-1 mRNA expression and addition of leupeptin, a protease inhibitor, to the cultures largely prevented the inhibition of MCP-1 expression by P. gingivalis. Transwell experiments showed that direct contact of P. gingivalis with THP-1 cells was not required for the MCP-1 inhibition. Furthermore, blockade of internalization of P. gingivalis into THP-1 cells had no effect on the MCP-1 inhibition by P. gingivalis. Finally, degradation of MCP-1 mRNA in LPS-stimulated THP-1 cells was accelerated in the presence of P. gingivalis. These results suggest that the proteolytic activity of P. gingivalis attenuate MCP-1 mRNA expression by promoting the decay of MCP-1 mRNA in LPS-stimulated THP-1 cells.

Anti-cardiolipin from periodontitis patients induces MCP-1 production by human umbilical vein endothelial cells.

AIM: Periodontal diseases are associated with a variety of systemic diseases, including cardiovascular disease and stroke, and patients with periodontitis demonstrate elevated levels of anti-cardiolipin antibodies. We sought to determine if anti-cardiolipin antibodies from periodontitis patients induced monocyte chemotactic protein-1 production by human vascular endothelial cells. MATERIALS AND METHODS: IgG was purified from sera from 53 subjects, including chronic and aggressive periodontitis patients and periodontally healthy controls, with elevated or normal IgG anti-cardiolipin levels. In addition, anti-cardiolipin antibodies were specifically removed from some sera by immunoabsorption. RESULTS: We found that, irrespective of diagnostic category, IgG from subjects with elevated anti-cardiolipin induced significantly greater monocyte chemotactic protein-1 production by human vascular endothelial cells than IgG from those subjects with normal anti-cardiolipin titres. Removal of anti-cardiolipin from IgG preparations from periodontitis patients significantly reduced their ability to induce monocyte chemotactic protein-1. CONCLUSIONS: Since elevated titres of anti-cardiolipin are found in a significantly greater proportion of patients with periodontitis than in periodontally healthy individuals, and these antibodies activate endothelial cells to produce monocyte chemotactic protein-1, they may explain some of the associations noted between periodontal infections and systemic conditions.

Flavonol-containing phosphorylated pullulan may attenuate pulp inflammation.

AIM: To find possible reagents to minimize inflammatory responses by using an established pulpitis models for the purpose of developing new pulp-capping materials, and to test the possible use of phosphorylated pullulan as a carrier for such an anti-inflammatory reagent. METHODOLOGY: Co-culturing was performed using transwell systems. Inflammatory responses were evaluated by measuring cytokines produced by the cells. The effects of two flavonoids, luteolin and quercetin, as anti-inflammatory reagents, and phosphorylated pullulan, which potentially achieves a sufficient marginal sealing to hydroxyapatite and slowly releases luteolin, as a carrier for flavonoids, were tested. RESULTS: Flavonols, particularly luteolin, dramatically attenuated inflammatory cytokine production, which was augmented by co-cultures. Luteolin was successfully enclosed by phosphorylated pullulan. Finally, it was confirmed that luteolin released from phosphorylated pullulan was effective in reducing cytokine production by co-cultures. CONCLUSIONS: Combination of phosphorylated pullulan and luteolin could be potentially used in the treatment of dental pulp inflammation.

Establishment of an ex vivo pulpitis model by co-culturing immortalized dental pulp cells and macrophages.

AIM: To establish an ex vivo pulpitis model by co-culturing dental pulp cells with macrophages. METHODOLOGY: As dental pulp cells, immortalized human dental pulp cells, named DP-1, were used, whilst as macrophage cell lines, the differentiated human monocytic cell line, THP-1, was used. In some experiments, primary dental pulp cells were isolated and used to confirm the results obtained in the experiments using immortalized cells. Co-culturing was performed using transwell systems. Inflammatory responses were evaluated by measuring cytokines produced by the cells. RESULTS: Co-culturing both cell types markedly up-regulated inflammatory cytokine production as compared with the cells cultured independently, suggesting that both cell types interact with each other to synergistically produce higher amounts of inflammatory cytokines. Interestingly, both DP-1 and primary dental pulp cells appeared to produce molecules stimulating macrophages to produce tumour necrosis factor-α-. CONCLUSION: Co-culturing immortalized dental pulp cells and macrophages may be a new ex vivo model for studying the pathophysiology of reversible pulpitis.

Peri-implantitis fibroblasts respond to host immune factor C1q.

BACKGROUND AND OBJECTIVE: Current therapies for peri-implantitis apply the same clinical protocols as those used for the treatment of periodontitis; however, outcomes remain unpredictable. We hypothesized that resident fibroblasts of the peri-implantitis stroma and periodontitis stroma differ in their phenotype and response to host immune factors. Fibroblasts are highly heterogeneous and comprise discrete subtypes with the potential of modulating inflammatory activities. The aim of the present study was to characterize the expression of receptors for complement C1q of innate immunity on human peri-implantitis fibroblasts and investigate effects of C1q on the proinflammatory properties of the cells. MATERIAL AND METHODS: Fibroblasts were cultured from gingival tissues exhibiting peri-implantitis and periodontitis, and from healthy gingivae as a control. Expression of C1q receptors for the collagen (cC1qR) and globular domains (gC1qR) of the protein was determined by flow cytofluorometric analysis (FITC) of specific antibodies bound to the surface of the cells. Secretion of C1q-inducible proinflammatory mediators was quantified after 24 h incubation using array-based ELISAs. RESULTS: The percentage of fibroblasts FITC-positive for cC1qR was 67, 75 and 12% in peri-implantitis, healthy and periodontitis cultures, respectively, whereas the percentage of gC1qR FITC-positive fibroblasts was 5, 3 and 59%, respectively. The C1q interactions with peri-implantitis and healthy fibroblasts increased secretion of the chemokines interleukin-6 and interleukin-8 twofold, and monocyte chemoattractant protein-1 fourfold over baseline values, whereas periodontitis fibroblasts were unresponsive. Complement C1q increased levels of vascular endothelial growth factor sevenfold and transforming growth factor-ß1 12-fold over baseline values in peri-implantitis cultures, only. CONCLUSIONS: Peri-implantitis fibroblasts differ from periodontitis fibroblasts in phenotypic expression of cC1qR and function, and from healthy fibroblasts in proinflammatory, angiogenic and fibrogenic function. Peri-implantitis fibroblasts may represent a novel subtype.

Alendronate regulates cytokine production induced by lipid A through nuclear factor-κB and Smad3 activation in human gingival fibroblasts.

BACKGROUND AND OBJECTIVE: Nitrogen-containing bisphosphonates (NBPs) are widely used as anti-bone-resorptive drugs. However, use of NBPs results in inflammatory side-effects, including jaw osteomyelitis. In the present study, we examined the effects of alendronate, a typical NBP, on cytokine production by human peripheral blood mononuclear cells (PBMCs) and gingival fibroblasts incubated with lipid A. METHODS: The PBMCs and gingival fibroblasts were pretreated with or without alendronate for 24 h. Cells were then incubated in the presence or absence of lipid A for a further 24 h. Levels of secreted human interleukin (IL)-1ß, IL-6, IL-8 and monocyte chemoattractant protein-1 (MCP-1) in culture supernatants were measured by ELISA. We also examined nuclear factor-κB (NF-κB) activation in both types of cells by ELISA. Activation of Smad3 in the cells was assessed by flow cytometry. In addition, we performed an inhibition assay using SIS3, a specific inhibitor for Smad3. RESULTS: Pretreatment of PBMCs with alendronate promoted lipid A-induced production of IL-1ß and IL-6, but decreased lipid A-induced IL-8 and MCP-1 production. In human gingival fibroblasts, alendronate pretreatment increased lipid A-induced production of IL-6 and IL-8, and increased NF-κB activation in gingival fibroblasts but not PBMCs stimulated with lipid A. In contrast, alendronate activated Smad3 in both types of cells. Finally, SIS3 inhibited alendronate-augmented IL-6 and IL-8 production by human gingival fibroblasts but up-regulated alendronate-decreased IL-8 production by PBMCs. CONCLUSION: These results suggest that alendronate-mediated changes in cytokine production by gingival fibroblasts occur via regulation of NF-κB and Smad3 activity.

Differential regulation of chemokine expression by estrogen in human periodontal ligament cells.

BACKGROUND AND OBJECTIVE: Estrogen modulates inflammatory responses, but the mechanisms involved have not yet been identified. Periodontal ligament (PDL) cells produce chemokines (a group of chemoattractant molecules that recruit leukocytes) and it has been suggested that estrogen modulates periodontal inflammation by regulating the expression of chemokines by PDL cells. Therefore, the objectives of this study were to investigate the regulation of chemokine ligand 2 [CCL2/monocyte chemoattractant protein 1 (MCP-1)], chemokine ligand 3 [CCL3/macrophage inflammatory protein-1α (MIP-1α)] and chemokine ligand 5 (CCL5/RANTES) by estrogen in human PDL cells. MATERIAL AND METHODS: PDL cells were obtained from the PDL of premolars, extracted for orthodontic reasons, from two boys and two girls (16 and 17 years of age). PDL cell CCL2, CCL3 and CCL5 mRNA transcripts were determined by quantitative real-time PCR. The concentrations of CCL2, CCL3 and CCL5 proteins were determined by ELISAs. RESULTS: Treatment with 0.5 µg/mL of lipopolysaccharide (LPS, from Escherichia coli) + 100 nm 17ß-estradiol (E(2) ) for 24 h reduced the expression of CCL3 mRNA by about 40% compared to PDL cells treated with LPS alone. Attenuation of CCL3 mRNA was not associated with a decrease in CCL3 protein within 48 h, suggesting a slow turnover of the CCL3 protein. Interindividual differences in the effects of E(2) on CCL5 mRNA expression were observed. E(2) (100 nm) increased the expression of CCL5 by 40-60% in PDL cells derived from two subjects but reduced the expression of CCL5 by about 30% in cells from another subject. CCL2 mRNA and CCL2 protein were highly expressed, but not regulated by E(2) . Similar data were observed in cells obtained from both boys and girls. CONCLUSION: Regulation, by estrogen, of chemokine expression in PDL cells shows a complex pattern involving the down-regulation as well as the up-regulation of chemokines, suggesting that estrogen exerts both anti-inflammatory and proinflammatory effects through these mechanisms.

Effects of oral commensal and pathogenic bacteria on human dendritic cells.

BACKGROUND/AIMS: The oral cavity harbors a diverse and complex microbial community. Bacteria accumulate on both the hard and soft oral tissues in sessile biofilms and engage the host in an intricate cellular dialog, which normally constrains the bacteria to a state of commensal harmony. Dendritic cells (DCs) are likely to balance tolerance and active immunity to commensal microorganisms as part of chronic inflammatory responses. While the role played by DCs in maintaining intestinal homeostasis has been investigated extensively, relatively little is known about DC responses to oral bacteria. METHODS: In this study, we pulsed human monocyte-derived immature DCs (iDCs) with cell wall extracts from pathogenic and commensal gram-positive or gram-negative oral bacteria. RESULTS: Although all bacterial extracts tested induced iDCs to mature and produce cytokines/chemokines including interleukin-12p40, tumor necrosis factor-alpha, and monocyte chemoattractant protein-1 (MCP-1), the most important factor for programming DCs by oral bacteria was whether they were gram-positive or gram-negative, not whether they were commensal or pathogenic. In general, gram-negative oral bacteria, except for periodontopathic Porphyromonas gingivalis, stimulated DC maturation and cytokine production at lower concentrations than gram-positive oral bacteria. The threshold of bacteria needed to stimulate chemokine production was 100-fold to 1000-fold lower than that needed to induce cytokines. In addition, very low doses of oral commensal bacteria triggered monocytes to migrate toward DC-derived MCP-1. CONCLUSION: Oral commensal and pathogenic bacteria do not differ qualitatively in how they program DCs. DC-derived MCP-1 induced in response to oral commensal bacteria may play a role, at least in part, in the maintenance of oral tissue integrity by attracting monocytes.

Monocyte chemotactic protein-1 mediates prostate cancer-induced bone resorption.

Prostate cancer preferentially metastasizes to bone, resulting in high mortality. Strategies to inhibit prostate cancer metastasis include targeting both tumor-induced osteoblastic lesions and underlying osteoclastic activities. We and others have previously shown that blocking receptor activator of nuclear factor-kappaB ligand (RANKL) partially blocks tumor establishment and progression in bone in murine models. However, levels of RANKL in the cell lines used in these studies were very low, suggesting that soluble factors other than RANKL may mediate the cancer-induced osteoclast activity. To identify these factors, a human cytokine antibody array was used to measure cytokine expression in conditioned medium collected from primary prostate epithelial cells (PrEC), prostate cancer LNCaP and its derivative C4-2B, and PC3 cells. All prostate cancer cells produced high amounts of monocyte chemotactic protein-1 (MCP-1) compared with PrEC cells. Furthermore, levels of interleukin (IL)-6, IL-8, GROalpha, ENA-78, and CXCL-16 were higher in PC3 than LNCaP. These results were confirmed by ELISA. Finally, human bone marrow mononuclear cells (HBMC) were cultured with PC3 conditioned medium. Although both recombinant human MCP-1 and IL-8 directly stimulated HBMC differentiation into osteoclast-like cells, IL-8, but not MCP-1, induced bone resorption on dentin slices with 21 days of culture in the absence of RANKL. However, the conditioned medium-induced bone resorption was inhibited by MCP-1 neutralizing antibody and was further synergistically inhibited with IL-8 antibody, indicating that MCP-1, in addition to IL-8, mediates tumor-induced osteoclastogenesis and bone resorption. MCP-1 may promote preosteoclast cell fusion, forming multinucleated tartrate-resistant acid phosphatase-positive osteoclast-like cells. This study may provide novel therapeutic targets for treatment of prostate cancer skeletal metastasis.

Expression of endothelial monocyte-activating polypeptide II in the rat dental follicle and its potential role in tooth eruption.

Endothelial monocyte-activating polypeptide II (EMAP-II) is an inflammatory cytokine with chemotactic activity. Because the dental follicle (DF) recruits mononuclear cells (osteoclast precursors) to promote the osteoclastogenesis needed for tooth eruption, it was the aim of this study to determine if EMAP-II contributes to this recruitment. Using a DNA microarray, EMAP-II was found to be highly expressed in vivo in the DFs of day 1 to day 11 postnatal rats, with its expression elevated on days 1 and 3. Use of a short interfering RNA (siRNA) to knock down EMAP-II expression resulted in a reduction in the expression of colony-stimulating factor-1 (CSF-1) and monocyte chemoattractant protein-1 (MCP-1) in the DF cells. Addition of EMAP-II protein to the DF cells partially restored the expression of CSF-1 and MCP-1. In chemotaxis assays using either conditioned medium of the DF cells with anti-(EMAP-II) immunoglobulin G added or conditioned medium of DF cells with EMAP-II knocked down by siRNA, migration indexes of bone marrow mononuclear cells were significantly reduced. These results suggest that EMAP-II is another chemotactic molecule in the dental follicle involved in the recruitment of mononuclear cells, and that EMAP-II may exert its chemotactic function directly by recruiting mononuclear cells and indirectly by enhancing the expression of other chemotactic molecules (CSF-1 and MCP-1).

In vitro induction of hydrolytic activity in human gingival and pulp fibroblasts by triethylene glycol dimethacrylate and monocyte chemotatic protein-1.

OBJECTIVE: Some dental resin composite materials leach the monomer triethylene glycol dimethacrylate (TEGDMA), which could contribute to the inflammatory reaction. The purpose of this study was to examine the ability of TEGDMA to alter cytokine/growth factor secretion and enzymatic activity in monocyte derived macrophages (U937 cells), human gingival fibroblasts (HGFs), and human pulp fibroblasts (DPFs). METHODS: A human growth factor/cytokine antibody array was utilized to determine whether TEGDMA alters the expression of cytokines/growth factors from U937 cells, HGFs, and DPFs. To determine if TEGDMA alters the hydrolase activity of U937, DPF, and HGF cells, the hydrolysis of p-nitrophenyl butyrate was utilized in a spectrophotometric assay. RESULTS: TEGDMA exposure induced the expression of monocyte chemotatic protein-1 (MCP-1) from the U937 cells. Both the cell lysates and conditioned media from the HGFs showed increased hydrolase activity after exposure to a sublethal concentration of TEGDMA. MCP-1 alone increased the hydrolytic activity and the combination of MCP-1 and TEGDMA was additive in HGF conditioned media. The DPFs were also exposed to MCP-1 alone and followed by a sublethal concentration of TEGDMA. The effect of MCP-1 was greater than that of TEGDMA. SIGNIFICANCE: These results demonstrate that TEGDMA induces enzymatic activity and cytokine/growth factor expression in a cell-specific manner.