Primary Human Peripheral Blood Monocytes Research Articles

Enamel matrix derivative does not affect osteoclast formation or bone resorption in cultures of mouse bone marrow macrophages or human monocytes

Objective Enamel matrix derivative (EMD) is widely used under the brand name Emdogain® to promote periodontal regeneration in surgical treatment of periodontitis and peri-implantitis. The molecular mechanisms are unclear, but it has been proposed that EMD has stimulatory effects on the root cementum and periodontal ligament cells. Since dental implants lack these structures, we hypothesized that EMD-induced bone gain involve interactions with osteoclast precursor cells, with consequent inhibitory effect on osteoclast formation and/or activity. The aim was to evaluate this hypothesis. Material and methods Primary mouse bone marrow macrophages (BMMs) and human peripheral blood monocytes were cultured in the presence of receptor activator nuclear factor-κB ligand (RANKL) to stimulate osteoclast formation. A purified Emdogain® fraction was added to the cell cultures and the effect on number and size of newly formed osteoclasts were evaluated. In cultures on natural bone slices, bioanalytical methods were used to assay osteoclast number and bone resorption. Results EMD had a negative effect on osteoclastogenesis in mouse cultures on plastic surface, whereas addition of EMD to osteoclast precursor cells on bone substrate did not affect osteoclast formation or bone resorption. Conclusions The results on natural bone matrix contradict a direct effect of EMD on osteoclast precursor cells.

Human Lung-Resident Macrophages Express and Are Targets of Thymic Stromal Lymphopoietin in the Tumor Microenvironment.

Thymic stromal lymphopoietin (TSLP) is a pleiotropic cytokine highly expressed by epithelial cells and several innate and adaptive immune cells. TSLP exerts its biological effects by binding to a heterodimeric complex composed of TSLP receptor (TSLPR) and IL-7Rα. In humans, there are two TSLP isoforms: the short form (sfTSLP), constitutively expressed, and the long form (lfTSLP), which is upregulated in inflammation. TSLP has been implicated in the induction and progression of several experimental and human cancers. Primary human lung macrophages (HLMs), monocyte-derived macrophages (MDMs), and peripheral blood monocytes consitutively expressed sfTSLP mRNA. Incubation of HLMs, MDMs, and monocytes with lipopolysaccharide (LPS) or IL-4, but not with IL-13, induced TSLP release from HLMs. LPS, but not IL-4 or IL-13, induced CXCL8 release from HLMs. LPS, IL-4 alone or in combination with IL-13, induced the expression of lfTSLP, but not of sfTSLP from HLMs. Preincubation of HLMs with IL-4, alone or in combination with IL-13, but not IL-13 alone, synergistically enhanced TSLP release from LPS-activated macrophages. By contrast, IL-4, alone or in combination with IL-13, inhibited LPS-induced CXCL8 release from HLMs. Immunoreactive TSLP was detected in lysates of HLMs, MDMs, and monocytes. Incubation of HLMs with TSLP induced the release of proinflammatory (TNF-α), angiogenic (VEGF-A, angiopoietin 2), and lymphangiogenic (VEGF-C) factors. TSLP, TSLPR, and IL-7Rα were expressed in intratumoral and peritumoral areas of human lung cancer. sfTSLP and lfTSLP mRNAs were differentially expressed in peritumoral and intratumoral lung cancer tissues. The TSLP system, expressed in HLMs, MDMs, and monocytes, could play a role in chronic inflammatory disorders including lung cancer.

A PKCβ-LYN-PYK2 Signaling Axis Is Critical for MCP-1-Dependent Migration and Adhesion of Monocytes.

MCP-1-induced monocyte chemotaxis is a crucial event in inflammation and atherogenesis. Identifying the important signal transduction pathways that control monocyte chemotaxis can unravel potential targets for preventive therapies in inflammatory disease conditions. Previous studies have shown that the focal adhesion kinase Pyk2 plays a critical role in monocyte motility. In this study, we investigated the MCP-1-mediated activation of Pyk2 (particularly by the phosphorylation of Tyr402) in primary human peripheral blood monocytes. We showed that MCP-1 induces Src phosphorylation in a similar time frame and that the MCP-1-induced Pyk2 tyrosine phosphorylation is controlled by the Src family kinase. We also report, in this study, that PKCβ, an isoform of PKC, is required for both Src and Pyk2 activation/phosphorylation in response to MCP-1 stimulation. We identified Lyn as the specific Src kinase isoform that is activated by MCP-1 and acts upstream of Pyk2 in primary monocytes. Furthermore, Lyn is found to be indispensable for monocyte migration in response to MCP-1 stimulation. Moreover, our coimmunoprecipitation studies in monocytes revealed that PKCβ, Pyk2, and Lyn exist constitutively in a molecular complex. To our knowledge, our study has uncovered a novel PKCβ-Lyn-Pyk2 signaling cascade in primary monocytes that regulates MCP-1-induced monocyte adhesion and migration.

HDAC1 and HDAC2 regulate anti-inflammatory effects of anesthetic isoflurane in human monocytes.

Pre-exposure to volatile anesthetics inhibits inflammation induced by various stimuli, including surgical procedures and ischemia. We hypothesize that volatile anesthetics may induce anti-inflammatory effects via a mechanism involving regulation of histone deacetylases (HDACs). Pre-exposure of 1.5% isoflurane for 0.5h induced anti-inflammatory effects [measured by cytokine production of tumor necrosis factor-ɑ, interleukin-8 (IL-8) and IL-1β] in both human THP-1 cells and primary human peripheral blood monocytes stimulated by lipopolysaccharide. In human THP-1 cells, coadministration of the HDAC inhibitor trichostatin A (TSA) blocked the isoflurane-induced anti-inflammatory effects. TSA also blocked isoflurane-upregulated HDAC1-3 expression and isoflurane-reduced nuclear translocation of p65 and p50 subunits of nuclear factor-κB (NF-κB). The ability of isoflurane to reduce NF-κB nuclear translocation and proinflammatory responses in the cell line was blocked by gene silencing of HDAC1 and HDAC2, but not by gene silencing of HDAC3. A coimmunoprecipitation assay demonstrated that the decreased interaction between HDAC1 and HDAC2 through lipopolysaccharide was restored by isoflurane pretreatment. These findings were validated in primary human peripheral blood monocytes wherein gene silencing of HDAC1 and HDAC2 resulted in increased cytokine production and NF-κB nuclear translocation induced by isoflurane pre-exposure and lipopolysaccharide stimulation. These results indicate that anti-inflammatory effects of the volatile anesthetic isoflurane in human monocytes involve regulation of HDAC1 and HDAC2.

Osteoclasts degrade fibrinogen scaffolds and induce mesenchymal stem/stromal osteogenic differentiation.

Fibrinogen (Fg) is a pro-inflammatory protein with pro-healing properties. Previous work showed that fibrinogen 3D scaffolds (Fg-3D) promote bone regeneration, but the cellular players were not identified. Osteoclasts are bone resorbing cells that promote bone remodeling in close crosstalk with osteoblasts. Herein, the capacity of osteoclasts differentiated on Fg-3D to degrade the scaffolds and promote osteoblast differentiation was evaluated in vitro. Fg-3D scaffolds were prepared by freeze-drying and osteoclasts were differentiated from primary human peripheral blood monocytes. Results obtained showed osteoclasts expressing the enzymes cathepsin K and tartrate resistant acid phosphatase colonizing Fg-3D scaffolds. Osteoclasts were able to significantly degrade Fg-3D, reducing the scaffold's area, and increasing D-dimer concentration, a Fg degradation product, in their culture media. Osteoclast conditioned media from the first week of differentiation promoted significantly stronger human primary mesenchymal stem/stromal cell (MSC) osteogenic differentiation, evaluated by alkaline phosphatase activity. Moreover, week 1 osteoclast conditioned media promoted earlier MSC osteogenic differentiation, than chemical osteogenesis inductors. TGF-β1 was found increased in osteoclast conditioned media from week 1, when compared to week 3 of differentiation. Taken together, our results suggest that osteoclasts are able to differentiate and degrade Fg-3D, producing factors like TGF-β1 that promote MSC osteogenic differentiation.

Toxoplasma gondii activates a Syk-CARD9-NF-κB signaling axis and gasdermin D-independent release of IL-1β during infection of primary human monocytes.

IL-1β is a potent pro-inflammatory cytokine that promotes immunity and host defense, and its dysregulation is associated with immune pathology. Toxoplasma gondii infection of myeloid cells triggers the production and release of IL-1β; however, the mechanisms regulating this pathway, particularly in human immune cells, are incompletely understood. We have identified a novel pathway of T. gondii induction of IL-1β via a Syk-CARD9-NF-κB signaling axis in primary human peripheral blood monocytes. Syk was rapidly phosphorylated during T. gondii infection of primary monocytes, and inhibiting Syk with the pharmacological inhibitors R406 or entospletinib, or genetic ablation of Syk in THP-1 cells, reduced IL-1β release. Inhibition of Syk in primary cells or deletion of Syk in THP-1 cells decreased parasite-induced IL-1β transcripts and the production of pro-IL-1β. Furthermore, inhibition of PKCδ, CARD9/MALT-1 and IKK reduced p65 phosphorylation and pro-IL-1β production in T. gondii-infected primary monocytes, and genetic knockout of PKCδ or CARD9 in THP-1 cells also reduced pro-IL-1β protein levels and IL-1β release during T. gondii infection, indicating that Syk functions upstream of this NF-κB-dependent signaling pathway for IL-1β transcriptional activation. IL-1β release from T. gondii-infected primary human monocytes required the NLRP3-caspase-1 inflammasome, but interestingly, was independent of gasdermin D (GSDMD) cleavage and pyroptosis. Moreover, GSDMD knockout THP-1 cells released comparable amounts of IL-1β to wild-type THP-1 cells after T. gondii infection. Taken together, our data indicate that T. gondii induces a Syk-CARD9/MALT-1-NF-κB signaling pathway and activation of the NLRP3 inflammasome for the release of IL-1β in a cell death- and GSDMD-independent manner. This research expands our understanding of the molecular basis for human innate immune regulation of inflammation and host defense during parasite infection.

Toxoplasma gondii disrupts β1 integrin signaling and focal adhesion formation during monocyte hypermotility

The motility of blood monocytes is orchestrated by the activity of cell-surface integrins, which translate extracellular signals into cytoskeletal changes to mediate adhesion and migration. Toxoplasma gondii is an intracellular parasite that infects migratory cells and enhances their motility, but the mechanisms underlying T. gondii-induced hypermotility are incompletely understood. We investigated the molecular basis for the hypermotility of primary human peripheral blood monocytes and THP-1 cells infected with T. gondii Compared with uninfected monocytes, T. gondii infection of monocytes reduced cell spreading and the number of activated β1 integrin clusters in contact with fibronectin during settling, an effect not observed in monocytes treated with lipopolysaccharide (LPS) or Escherichia coli Furthermore, T. gondii infection disrupted the phosphorylation of focal adhesion kinase (FAK) at tyrosine 397 (Tyr-397) and Tyr-925 and of the related protein proline-rich tyrosine kinase (Pyk2) at Tyr-402. The localization of paxillin, FAK, and vinculin to focal adhesions and the colocalization of these proteins with activated β1 integrins were also impaired in T. gondii-infected monocytes. Using time-lapse confocal microscopy of THP-1 cells expressing enhanced GFP (eGFP)-FAK during settling on fibronectin, we found that T. gondii-induced monocyte hypermotility was characterized by a reduced number of enhanced GFP-FAK-containing clusters over time compared with uninfected cells. This study demonstrates an integrin conformation-independent regulation of the β1 integrin adhesion pathway, providing further insight into the molecular mechanism of T. gondii-induced monocyte hypermotility.

NLRP3 and Potassium Efflux Drive Rapid IL-1β Release from Primary Human Monocytes during Toxoplasma gondii Infection.

IL-1β is produced by myeloid cells and acts as a critical mediator of host defense during infection and injury. We found that the intracellular protozoan parasite Toxoplasma gondii induced an early IL-1β response (within 4 h) in primary human peripheral blood monocytes isolated from healthy donors. This process involved upregulation of IL-1β, IL-1RN (IL-1R antagonist), and NLRP3 transcripts, de novo protein synthesis, and the release of pro- and mature IL-1β from infected primary monocytes. The released pro-IL-1β was cleavable to mature bioactive IL-1β in the extracellular space by the protease caspase-1. Treatment of primary monocytes with the NLRP3 inhibitor MCC950 or with extracellular potassium significantly reduced IL-1β cleavage and release in response to T. gondii infection, without affecting the release of TNF-α, and indicated a role for the inflammasome sensor NLRP3 and for potassium efflux in T. gondii-induced IL-1β production. Interestingly, T. gondii infection did not induce an IL-1β response in primary human macrophages derived from the same blood donors as the monocytes. Consistent with this finding, NLRP3 was downregulated during the differentiation of monocytes to macrophages and was not induced in macrophages during T. gondii infection. To our knowledge, these findings are the first to identify NLRP3 as an inflammasome sensor for T. gondii in primary human peripheral blood cells and to define an upstream regulator of its activation through the release of intracellular potassium.

The interaction between acetylation and serine-574 phosphorylation regulates the apoptotic function of FOXO3.

The multispecific transcription factor and tumor suppressor FOXO3 is an important mediator of apoptosis, but the mechanisms that control its proapoptotic function are poorly understood. There has long been evidence that acetylation promotes FOXO3-driven apoptosis and recently a specific JNK (c-Jun N-terminal kinase)-dependent S574 phosphorylated form (p-FOXO3) has been shown to be specifically apoptotic. This study examined whether acetylation and S574 phosphorylation act independently or in concert to regulate the apoptotic function of FOXO3. We observed that both sirtuins 1 and 7 (SIRT1 and SIRT7) are able to deacetylate FOXO3 in vitro and in vivo, and that lipopolysaccharide (LPS) treatment of THP-1 monocytes induced a rapid increase of FOXO3 acetylation, partly by suppression of SIRT1 and SIRT7. Acetylation was required for S574 phosphorylation and cellular apoptosis. Deacetylation of FOXO3 by SIRT activation or SIRT1 or SIRT7 overexpression prevented its S574 phosphorylation and blocked apoptosis in response to LPS. We also found that acetylated FOXO3 preferentially bound JNK1, and a mutant FOXO3 lacking four known acetylation sites (K242, 259, 290 and 569R) abolished JNK1 binding and failed to induce apoptosis. This interplay of acetylation and phosphorylation also regulated cell death in primary human peripheral blood monocytes (PBMs). PBMs isolated from alcoholic hepatitis patients had high expression of SIRT1 and SIRT7 and failed to induce p-FOXO3 and apoptosis in response to LPS. PBMs from healthy controls had lower SIRT1 and SIRT7 and readily formed p-FOXO3 and underwent apoptosis when similarly treated. These results reveal that acetylation is permissive for generation of the apoptotic form of FOXO3 and the activity of SIRT1 and particularly SIRT7 regulate this process in vivo, allowing control of monocyte apoptosis in response to LPS.

Membrane trafficking and PIP2‐dependent regulation of Kir2.2 by LPS in THP‐1 human monocyte (LB839)

Lipopolysaccharide (LPS) is an effective agonist for Toll‐like receptor 4 (TLR4) in monocyte/macrophage. However, investigation on ion channels affected by LPS is not completely understood. In THP‐1 human monocyte cells, we observed decrease in voltage‐gated K+ channel current (51 %) and increase in Ca2+‐activated K+ channel (235 %) at 6 h after LPS application (1 μg/ml). Interestingly, marked increase of inwardly rectifying K+ channel (Kir) current was observed from 1‐2 h (IKir‐LPS), which peaked at 4 h and spontaneously decayed to 27 % of peak at 24 h. Single channel conductance of the IKir‐LPS was 38 pS, and their Ba2+‐sensitivity was relatively high (IC50, 1.42 μM), which were consistent with Kir2.2 isotype. Functional upregulation of IKir‐LPS was also found in primary human peripheral blood monocytes. While total Kir2.2 protein was not changed, its membrane fraction was increased time‐dependently and confocal microscopy confirmed the increased plasma membrane expression from 2 h. Also, IKir‐LPS was partially attenuated by Exo‐1 (vesicular trafficking inhibitor). However, the spontaneous decay of IKir‐LPS after 4 h was not prevented by dynamin inhibitor (Dynasore). Interestingly, the decayed IKir‐LPS at 24 h was rapidly reversed by PI3 kinase (PI3K) inhibitors (Wortmannin and LY294002). In contrast, IKir‐LPS was suppressed by bpV(Phen), an inhibitor for PTEN (PIP3 phosphatase). IKir‐LPS was not affected by Akt inhibitors, suggesting that the decay of IKir‐LPS was not due to PIP3‐dependent signaling but to decreased availability of PIP2 (conversion to PIP3 by PI3K) that is known to be critical for Kir2.2 activity. Store‐operated Ca2+ entry significantly increased in 4 h LPS treated THP‐1, which was prevented by KCl‐induced depolarization. Taken together, we firstly report that Kir2.2 is functionally upregulated by LPS via membrane trafficking in human monocytes. Subsequent hyperpolarization enhanced Ca2+ influx, implying a physiological role of K+ channels in the innate immunity.

On the role of 25-hydroxycholesterol synthesis by glioblastoma cell lines. Implications for chemotactic monocyte recruitment

Glioblastoma multiforme (GBM) is the most common malignant primary brain tumor and is invariably fatal to affected patients. Oxysterols belong to a class of bioactive lipids that are implicated in neurological disease and are associated with various types of cancer. Here, we investigated expression and transcriptional regulation of cholesterol 25-hydroxylase (CH25H) in human U87MG and GM133 glioblastoma cell lines. We demonstrate that in both cell lines transcription and translation of CH25H are increased in response to TNFα and IL1β. In parallel, both cell lines upregulate 25-hydroxycholesterol (25-OHC) synthesis and secretion to levels comparable to bone marrow-derived mouse macrophages under inflammatory conditions. To determine whether 25-OHC acts as chemoattractant for tumor-associated macrophages, the human THP-1 monoblastic leukemia cell line was treated with varying amounts of the oxysterol. Experiments revealed that 25-OHC and lipid extracts isolated from GM133-conditioned medium (containing 7-fold higher 25-OHC concentrations than U87MG medium) induce chemotactic migration of THP-1 cells. Of note, 25-OHC also induced the migration of primary human peripheral blood monocytes. In response to exogenously added 25-OHC, THP-1 cells reorganized intermediate filament-associated vimentin to more cortical and polarized structures. Chemotactic migration of monocytes in response to 25-OHC was pertussis toxin-sensitive, indicating the involvement of G protein-coupled receptors. Using RNA interference we demonstrated that G protein–coupled receptor 183 (EBI2) contributes to 25-OHC-mediated chemotactic migration of THP-1 cells. These in vitro data indicate that GBM-derived and secreted 25-OHC may be involved in the recruitment of immune-competent cells to a tumor via EBI2.

Fibrinogen promotes resorption of chitosan by human osteoclasts

The osteoconductive and osteoinductive properties of materials intended for bone regeneration have been extensively tested, but the resorbability of these materials is often overlooked. Osteoclasts are responsible for bone resorption and play a crucial role in bone remodeling, which is essential for complete regeneration of bone tissue following injury. In this study we compare, for the first time, the ability of unmodified and fibrinogen (Fg)-modified chitosan (Ch) substrates to support the formation of multinucleated osteoclasts, and the potential of these cells to resorb the two substrates in vitro. Osteoclasts were differentiated from primary human peripheral blood monocytes directly on the substrates being investigated. Our results showed similar cell adhesion to unmodified and Fg-modified Ch substrates. Although the number of multinucleated osteoclasts on both Ch substrates increased throughout the culture period, by 21days of culture significantly more highly multinucleated osteoclasts (>10 nuclei per cell) were observed on Fg-modified Ch, when compared to Ch alone. In addition, cells were tartrate-resistant acid phosphatase positive and secreted significantly more enzyme on Ch-based substrates than in control conditions. Unmodified and Fg-modified Ch resorption was investigated by fluorescence microscopy and confirmed by electron microscopy. Quantification of results obtained by fluorescence microscopy shows that Fg modification led to significantly higher substrate resorption by 17days of culture. Our results show that osteoclasts, beyond resorbing mineralized substrates, successfully resorb a polymeric substrate (Ch), with Fg accelerating this process. Thus, in bone tissue regeneration strategies employing polymeric biomaterials, resorption may depend not only on macrophages, but also on osteoclasts.

The HIV-1 gp120/V3 modifies the response of uninfected CD4 T cells to antigen presentation: mapping of the specific transcriptional signature

BackgroundThe asymptomatic phase of HIV-1 infection is characterized by a progressive depletion of uninfected peripheral effector/memory CD4+ T cells that subsequently leads to immune dysfunction and AIDS symptoms. We have previously demonstrated that the presence of specific gp120/V3 peptides during antigen presentation can modify the activation of normal T-cells leading to altered immune function. The aim of the present study was to map the specific transcriptional profile invoked by an HIV-1/V3 epitope in uninfected T cells during antigen presentation.MethodsWe exposed primary human peripheral blood monocytes to V3 lipopeptides using a liposome delivery system followed by a superantigen-mediated antigen presentation system. We then evaluated the changes in the T-cell transcriptional profile using oligonucleotide microarrays and performed Ingenuity Pathway Analysis (IPA) and DAVID analysis. The results were validated using realtime PCR, FACS, Western blotting and immunofluorescence.ResultsOur results revealed that the most highly modulated transcripts could almost entirely be categorized as related to the cell cycle or transcriptional regulation. The most statistically significant enriched categories and networks identified by IPA were associated with cell cycle, gene expression, immune response, infection mechanisms, cellular growth, proliferation and antigen presentation. Canonical pathways involved in energy and cell cycle regulation, and in the co-activation of T cells were also enriched.ConclusionsTaken together, these results document a distinct transcriptional profile invoked by the HIV-1/V3 epitope. These data could be invaluable to determine the underlying mechanism by which HIV-1 epitopes interfere with uninfected CD4+ T-cell function causing hyper proliferation and AICD.

Monocyte-derived macrophages matured under prolonged hypoxia transcriptionally up-regulate HIF-1α mRNA

This study tested the hypothesis that prolonged severe hypoxia during monocyte to macrophage differentiation results in macrophages with a pattern of gene expression and phenotype distinct from those maturing in normal oxygen levels. Macrophages accumulate in hypoxic and anoxic areas within pathological sites such as tumours, wounds, and arthritic joints, and have been proposed as vehicles for gene therapy delivery to such tissues. Several non-pathological tissues are also hypoxic. We therefore argue that differentiation from monocyte to macrophage in hypoxic conditions is a common occurrence. However, the effect of long term severe hypoxia on monocyte to macrophage differentiation has not been studied. Here, using primary human peripheral blood monocytes, we show that maturation for 5 days in 0.2% oxygen results in decreased phagocytosis, and decreased CD40 and CD206 expression. Chronic hypoxia induced much higher mRNA levels of the pro-angiogenic cytokine, VEGF, in adherence-purified macrophages (27-fold), CD14-magnetic bead purified monocytes (90-fold), and PBMC (104-fold) compared to acute (24 h) hypoxia (11, 17 and 9-fold, respectively). This suggests that macrophages may play an even greater role in angiogenesis than previously appreciated. Furthermore, chronic hypoxia resulted in up-regulation of HIF-1α mRNA, in all monocyte-derived macrophage types studied. Actinomycin D experiments indicate that the increases in HIF-1α mRNA were not due to increased mRNA stability. To our knowledge this is the first study demonstrating up-regulation of HIF-1α mRNA by hypoxia in macrophages. Taken together, the data support the hypothesis that hypoxia affects monocyte to macrophage maturation, resulting in a distinct gene expression pattern and phenotype.

Direct Inhibition of Human RANK+ Osteoclast Precursors Identifies a Homeostatic Function of IL-1β

IL-1β is a key mediator of bone resorption in inflammatory settings, such as rheumatoid arthritis (RA). IL-1β promotes osteoclastogenesis by inducing RANKL expression on stromal cells and synergizing with RANKL to promote later stages of osteoclast differentiation. Because IL-1Rs share a cytosolic Toll-IL-1R domain and common intracellular signaling molecules with TLRs that can directly inhibit early steps of human osteoclast differentiation, we tested whether IL-1β also has suppressive properties on osteoclastogenesis in primary human peripheral blood monocytes and RA synovial macrophages. Early addition of IL-1β, prior to or together with RANKL, strongly inhibited human osteoclastogenesis as assessed by generation of TRAP(+) multinucleated cells. IL-1β acted directly on human osteoclast precursors (OCPs) to strongly suppress expression of RANK, of the costimulatory triggering receptor expressed on myeloid cells 2 receptor, and of the B cell linker adaptor important for transmitting RANK-induced signals. Thus, IL-1β rendered early-stage human OCPs refractory to RANK stimulation. Similar inhibitory effects of IL-1β were observed using RA synovial macrophages. One mechanism of RANK inhibition was IL-1β-induced proteolytic shedding of the M-CSF receptor c-Fms that is required for RANK expression. These results identify a homeostatic function of IL-1β in suppressing early OCPs that contrasts with its well-established role in promoting later stages of osteoclast differentiation. Thus, the rate of IL-1-driven bone destruction in inflammatory diseases, such as RA, can be restrained by its direct inhibitory effects on early OCPs to limit the extent of inflammatory osteolysis.

Temporal Interleukin-1β Secretion from Primary Human Peripheral Blood Monocytes by P2X7-independent and P2X7-dependent Mechanisms

The processing and regulated secretion of IL-1β are critical points of control of the biological activity of this important pro-inflammatory cytokine. IL-1β is produced by both monocytes and macrophages, but the rate and mechanism of release differ according to the differentiation status and the origin of these cells. We aimed to study the control of processing and release in human blood monocytes and human monocyte-derived macrophages. Toll-like receptor (TLR)-induced IL-1β production and release were investigated for dependence upon caspase-1, P2X7 receptor activation, and loss of membrane asymmetry associated with microvesicle shedding. TLR agonists induced P2X7 receptor-dependent IL-1β release in both monocytes and macrophages; however, only monocytes also showed P2X7 receptor-independent release of mature IL-1β. Furthermore, in monocytes ATP-mediated PS exposure could be activated independently of IL-1β production. Release of IL-1β from monocytes showed selectivity for specific TLR agonists and was accelerated by P2X7 receptor activation. Human monocytes released more IL-1β/cell than macrophages. These data have important implications for inflammatory diseases that involve monocyte activation and IL-1 release.

CXC Chemokine Ligand 4 Induces a Unique Transcriptome in Monocyte-Derived Macrophages

In atherosclerotic arteries, blood monocytes differentiate to macrophages in the presence of growth factors, such as macrophage colony-stimulation factor (M-CSF), and chemokines, such as platelet factor 4 (CXCL4). To compare the gene expression signature of CXCL4-induced macrophages with M-CSF-induced macrophages or macrophages polarized with IFN-gamma/LPS (M1) or IL-4 (M2), we cultured primary human peripheral blood monocytes for 6 d. mRNA expression was measured by Affymetrix gene chips, and differences were analyzed by local pooled error test, profile of complex functionality, and gene set enrichment analysis. Three hundred seventy-five genes were differentially expressed between M-CSF- and CXCL4-induced macrophages; 206 of them overexpressed in CXCL4 macrophages coding for genes implicated in the inflammatory/immune response, Ag processing and presentation, and lipid metabolism. CXCL4-induced macrophages overexpressed some M1 and M2 genes and the corresponding cytokines at the protein level; however, their transcriptome clustered with neither M1 nor M2 transcriptomes. They almost completely lost the ability to phagocytose zymosan beads. Genes linked to atherosclerosis were not consistently upregulated or downregulated. Scavenger receptors showed lower and cholesterol efflux transporters showed higher expression in CXCL4- than M-CSF-induced macrophages, resulting in lower low-density lipoprotein content. We conclude that CXCL4 induces a unique macrophage transcriptome distinct from known macrophage types, defining a new macrophage differentiation that we propose to call M4.

Major surface protein complex ofTreponema denticolainduces the production of tumor necrosis factor α, interleukin-1β, interleukin-6 and matrix metalloproteinase 9 by primary human peripheral blood monocytes

Treponema denticola is a micro-organism that is involved in the pathogenesis of periodontitis. Major surface protein complex (MSPc), which is expressed on the envelope of this treponeme, plays a key role in the interaction between T. denticola and gingival cells. The peptidoglycan extracted from T. denticola induces the production of a large variety of inflammatory mediators by macrophage-like cells, suggesting that individual components of T. denticola cells induce the inflammatory response during periodontal disease. This study was designed to demonstrate that MSPc of T. denticola stimulates release of proinflammatory mediators in primary human monocytes. Primary human monocytes were separated from the blood of healthy donors and incubated for up to 24 h with varying concentrations of MSPc. The production of tumor necrosis factor alpha (TNF-alpha), interleukin-1 beta (IL-1 beta), interleukin-6 (IL-6) and matrix metalloproteinase 9 (MMP-9) was measured at different time points with commercially available enzyme-linked immunosorbent assays. T. denticola MSPc induced the synthesis of TNF-alpha, IL-1 beta, IL-6 and MMP-9 in a dose- and time-dependent manner. Similar patterns of TNF-alpha, IL-1 beta and IL-6 release were observed when cells were stimulated with 100 and 1000 ng/mL of MSPc. The production of MMP-9 was significant only when cells were treated with 1000 ng/mL of MSPc. These results indicate that T. denticola MSPc, at concentrations ranging from 100 ng/mL to 1.0 microg/mL, activates a proinflammatory response in primary human monocytes.

Galphas-coupled receptor signaling actively down-regulates α4β1-integrin affinity: A possible mechanism for cell de-adhesion

BackgroundActivation of integrins in response to inside-out signaling serves as a basis for leukocyte arrest on endothelium, and migration of immune cells. Integrin-dependent adhesion is controlled by the conformational state of the molecule (i.e. change in the affinity for the ligand and molecular unbending (extension)), which is regulated by seven-transmembrane Guanine nucleotide binding Protein-Coupled Receptors (GPCRs). α4β1-integrin (CD49d/CD29, Very Late Antigen-4, VLA-4) is expressed on leukocytes, hematopoietic stem cells, hematopoietic cancer cells, and others. Affinity and extension of VLA-4 are both rapidly up-regulated by inside-out signaling through several Gαi-coupled GPCRs. The goal of the current report was to study the effect of Gαs-coupled GPCRs upon integrin activation.ResultsUsing real-time fluorescent ligand binding to assess affinity and a FRET based assay to probe α4β1-integrin unbending, we show that two Gαs-coupled GPCRs (H2-histamine receptor and β2-adrenergic receptor) as well as several cAMP agonists can rapidly down modulate the affinity of VLA-4 activated through two Gαi-coupled receptors (CXCR4 and FPR) in U937 cells and primary human peripheral blood monocytes. This down-modulation can be blocked by receptor-specific antagonists. The Gαs-induced responses were not associated with changes in the expression level of the Gαi-coupled receptors. In contrast, the molecular unbending of VLA-4 was not significantly affected by Gαs-coupled GPCR signaling. In a VLA-4/VCAM-1-specific myeloid cell adhesion system, inhibition of the VLA-4 affinity change by Gαs-coupled GPCR had a statistically significant effect upon cell aggregation.ConclusionWe conclude that Gαs-coupled GPCRs can rapidly down modulate the affinity state of VLA-4 binding pocket through a cAMP dependent pathway. This plays an essential role in the regulation of cell adhesion. We discuss several possible implications of this described phenomenon.

IN VITRO MODELING OF HUMAN ALVEOLAR MACROPHAGE SMOKE EXPOSURE: ENHANCED INFLAMMATION AND IMPAIRED FUNCTION

Pulmonary macrophages (MØs) are essential for clearance of inhaled particles, innate immunity, and lung tissue maintenance. However, the products of activated MØs have also been implicated in inflammation and tissue destruction, including in chronic obstructive pulmonary disease (COPD). Primary human alveolar macrophages (AMs) are available in limited numbers via bronchoalveolar lavage (BAL) or sputum induction, and BAL macrophages are not commonly available to all researchers. A readily available, plentiful, but representative surrogate for AMs would advance understanding of the contribution of macrophages to lung pathophysiology. Herein the authors describe a method for the in vitro derivation of AM-like cells using primary human peripheral blood monocytes differentiated in suspension with granulocyte-macrophage colony-stimulating factor (GM-CSF). The method produces a cell population with a consistent and stable phenotype. Flow cytometry reveals that GM-CSF–derived macrophages (GM-MØs) express lineage markers, immunoglobulin gamma (Fcγ) receptors, adhesion molecules, antigen presentation coreceptors, and scavenger receptors akin to AMs. Functionally, cigarette smoke activates extracellular signal-related kinase (ERK) and p38 mitogen-activated protein (MAP) kinase, enhances interleukin 8 (IL8) production from GM-MØs and inhibits phagocytosis, phenotypes previously described for smokers' AMs. Global transcriptional profiling revealed significant overlap in regulated genes between smokers' AMs and GM-MØs treated with cigarette smoke preparations in vitro.