Mature Monocyte-derived Macrophages Research Articles

Anti-inflammatory effects of naproxen sodium on human osteoarthritis synovial fluid immune cells

To evaluate the anti-inflammatory effects of clinically relevant naproxen sodium (Nx) concentrations on human monocyte-derived macrophages in a controlled in vitro system and human primary synovial fluid (SF) cells. Using phorbol 12-myristate 13-acetate, THP-1 human monocytic cells were differentiated into mature monocyte-derived macrophages in vitro then treated with Nx pre- or post-activating an inflammatory response with lipopolysaccharide (LPS) and hyaluronan (HA) fragments (n=8/group). Cell culture supernatants were assessed for NF-κB activity and prostaglandin E2 (PGE2), indicating cyclooxygenase enzyme activity. Under Duke IRB approval, primary human SF cells were collected at the time of knee joint replacement (n=19 individuals) for osteoarthritis (OA), and cultured with LPS, HA and Nx; SF cells were characterized by polychromatic flow cytometry for cell surface markers and intracellular cytokines. Compared to placebo treatment of THP-1cells, low dose Nx (corresponding 27.5-440mg/L orally) added both pre- and post-activation with LPS/HA, significantly reduced NF-κB activity and PGE2: mean reduction to 73%, 61%, 17% and 10% of placebo, respectively. LPS/HA treatment of primary OA SF cells significantly increased the number of IL-1β producing primary monocytes and macrophages, and by 24h the overall production of secreted cytokines (IL-1β, IL-6, IL8, and TNF-α). Low dose Nx reduced the percentage of IL-1β producing primary monocytes and macrophages. LPS/HA induced inflammation of THP-1 monocytic and primary human SF cells. Low dose Nx both prevented and reduced inflammatory responses of a human monocytic cell line and reduced IL-1β production by primary human SF monocytes and macrophages.

Biliary apotopes and anti-mitochondrial antibodies activate innate immune responses in primary biliary cirrhosis

Our understanding of primary biliary cirrhosis (PBC) has been significantly enhanced by the rigorous dissection of the multilineage T and B cell response against the immunodominant mitochondrial autoantigen, the E2 component of the pyruvate dehydrogenase complex (PDC-E2). PDC-E2 is a ubiquitous protein present in mitochondria of nucleated cells. However, the damage of PBC is confined to small biliary epithelial cells (BECs). We have previously demonstrated that BECs translocate immunologically intact PDC-E2 to apoptotic bodies and create an apotope. To define the significance of this observation, we have studied the ability of biliary or control epithelial apotopes to induce cytokine secretion from mature monocyte-derived macrophages (MDMphis) from either patients with PBC or controls in the presence or absence of anti-mitochondrial antibodies (AMAs). We demonstrate that there is intense inflammatory cytokine production in the presence of the unique triad of BEC apotopes, macrophages from patients with PBC, and AMAs. The cytokine secretion is inhibited by anti-CD16 and is not due to differences in apotope uptake. Moreover, MDMphis from PBC patients cultured with BEC apoptotic bodies in the presence of AMAs markedly increase tumor necrosis factor-related apoptosis-inducing ligand expression. These results provide a mechanism for the biliary specificity of PBC, the recurrence of disease after liver transplantation, and the success of ursodiol in treatment. They further emphasize the critical role of the innate immune system in the perpetuation of this autoimmune disease.

556 POSTER Post-radiation reaction includes induction of fluoropyrimidine metabolizing enzymes – a concept supporting enhancement of capecitabine anabolism regardless of timing administration

In the present study we have explored the catabolism of dCyd and 2′,3′-dideoxycytidine (ddC) in mature monocyte-derived macrophages (M/M) and focused on its relation to the accumulation of intracellular anabolites. We found that dCyd catabolism in M/M was significant in 1-week old cultures (15% of 0.5 μM dCyd, 0.5 nmole/million cells, catabolized within one hour of incubation) and further increased in more mature 3-week and 5-week old cultures (>85% catabolized within one hour). Dihydrouracil (DHU) was irreversibly formed as end product of the dCyd catabolism, with dUrd and Ura as intermediate metabolites. Intracellularly, dCyd nucleotides were detected up to three hours of incubation, with dCTP maintained at steady levels between one and three hours. However, after twelve hours of incubation DHU was the only detectable intracellular as well as extracellular metabolite. In contrast to what was observed with dCyd, we found that ddC was resistant to catabolism, with no detectable catabolites formed within twenty-four hours of incubation. Therefore ddC anabolism was unopposed by catabolic pathways and consequently intracellular anabolites of ddC accumulated throughout twenty-four hours of incubation. Based on these results, we propose that lack (or inhibition) of catabolism is at least as important as efficient anabolic phosphorylation for obtaining therapeutic effects of pyrimidine nucleoside analogs.

Cyclic biaxial strain affects U937 macrophage‐like morphology and enzymatic activities

As monocytes migrate to the site of a foreign body and differentiate into mature monocyte-derived macrophages (MDMs), the cells undergo a morphological transformation that involves mechanical stimulation via membrane stretch. Because the site of many cardiovascular implant devices includes substrates that are also undergoing mechanical change, it is of interest to assess the effect of such dynamic conditions on cellular-biomaterial responses. This study investigated the influence of cyclic (0.25 Hz) biaxial strain (maximum 10% amplitude) on human U937 macrophage-like cells cultured on a flexible siloxane membrane. Cell attachment was unaffected by the strain but total protein levels were significantly higher in stimulated cells. Intracellular esterase and released acid phosphatase activities were elevated by dynamic loading in addition to a strain-induced increase of monocyte-specific esterase protein as demonstrated by immunoblotting analysis. The morphology of static cells changed with cyclic strain from a round cell shape to an irregular, spread phenotype with a progressive reorganization of filamentous actin. The focal adhesion protein vinculin showed distinct reorganization in structure going from a well-defined arrangement in static cells to a diffuse staining pattern in mechano-stimulated cells. This study has demonstrated that U937 cells respond to cyclic deformation with an augmentation of select enzymatic activities that have been identified as being important in polymer biodegradation processes, as well as morphological changes, which may be characteristic of mechanical stress-induced cell activation.

The human β‐glucan receptor is widely expressed and functionally equivalent to murine Dectin‐1 on primary cells

We identified the C-type-lectin-like receptor, Dectin-1, as the major receptor for fungal beta-glucans on murine macrophages and have demonstrated that it plays a significant role in the cellular response to these carbohydrates. Using two novel, isoform-specific mAb, we show here that human Dectin-1, the beta-glucan receptor (betaGR), is widely expressed and present on all monocyte populations as well as macrophages, DC, neutrophils and eosinophils. This receptor is also expressed on B cells and a subpopulation of T cells, demonstrating that human Dectin-1 is not myeloid restricted. Both major functional betaGR isoforms - betaGR-A and betaGR-B - were expressed by these cell populations in peripheral blood; however, only betaGR-B was significantly expressed on mature monocyte-derived macrophages and immature DC, suggesting cell-specific control of isoform expression. Inflammatory cells, recruited in vivo using a new skin-window technique, demonstrated that Dectin-1 expression was not significantly modulated on macrophages during inflammation, but is decreased on recruited granulocytes. Despite previous reports detailing the involvement of other beta-glucan receptors on mature human macrophages, we have demonstrated that Dectin-1 acted as the major beta-glucan receptor on these cells and contributed to the inflammatory response to these carbohydrates.

Electron microscope studies of the replication of a British isolate of maedi visna virus in macrophages and skin cell lines

The replication of EV1, a British isolate of maedi visna virus (MVV), in macrophages has not previously been studied. We therefore used transmission and scanning electron microscopy (TEM and SEM respectively) to compare the replication of EV1 in macrophages versus skin cell lines. Monocyte-derived macrophages (MDM), alveolar macrophages (AM) and skin cell lines were all permissive for replication by EV1. Virus grew rapidly and to high titers in skin cell lines and mature MDM. However replication was slower in less mature MDM or AM. Virion budding occurred through (i) cytoplasmic membranes only (skin cells), (ii) cytoplasmic and vesicular membranes (MDM) or (iii) vesicular membranes only (AM). This meant that virions accumulated in vacuoles within macrophages. Retroviral intracytoplasmic type A particles were seen in the cytoplasm of AM and MDM infected with strain EV1, but not MDM infected with strain 1514 (an Icelandic MVV strain) and were shown to contain MVV gag antigen by immunogold staining.

Change in sensitivity to lipopolysaccharide during the differentiation of human monocytes to macrophages in vitro.

Mononuclear phagocytes in distinct differentiation stages and cultured under different conditions were tested for their sensitivity towards lipopolysaccharide (LPS), using procoagulant activity (PCA) expression and tumor necrosis factor (TNF) production as indices. The response of mature monocyte-derived macrophages differed from that of freshly isolated monocytes 1) by higher levels of constitutive PCA, 2) by responding to approximately 1,000-fold lower concentrations of LPS with PCA and TNF production, and 3) by a faster rise in PCA and TNF production. Due to the high constitutive level of PCA expression, the PCA stimulation index for LPS was low in macrophages when compared with that in monocytes. Thus, during differentiation to macrophages, human monocytes acquire increased sensitivity to LPS (2 orders of magnitude more sensitive than a sensitive turbidimetric Limulus amoebocyte lysate assay). This exquisite sensitivity to LPS is expressed regardless of whether LPS is offered in the presence or absence of lipopolysaccharide binding protein-containing serum. This points to as yet uncharacterized pathways of high affinity interaction between LPS and macrophages.

Deoxycytidine and 2′,3′-Dideoxycytidine Metabolism in Human Monocyte-Derived Macrophages: A Study of Both Anabolic and Catabolic Pathways

In the present study we have explored the catabolism of dCyd and 2′,3′-dideoxycytidine (ddC) in mature monocyte-derived macrophages (M/M) and focused on its relation to the accumulation of intracellular anabolites. We found that dCyd catabolism in M/M was significant in 1-week old cultures (15% of 0.5 μM dCyd, 0.5 nmole/million cells, catabolized within one hour of incubation) and further increased in more mature 3-week and 5-week old cultures (>85% catabolized within one hour). Dihydrouracil (DHU) was irreversibly formed as end product of the dCyd catabolism, with dUrd and Ura as intermediate metabolites. Intracellularly, dCyd nucleotides were detected up to three hours of incubation, with dCTP maintained at steady levels between one and three hours. However, after twelve hours of incubation DHU was the only detectable intracellular as well as extracellular metabolite. In contrast to what was observed with dCyd, we found that ddC was resistant to catabolism, with no detectable catabolites formed within twenty-four hours of incubation. Therefore ddC anabolism was unopposed by catabolic pathways and consequently intracellular anabolites of ddC accumulated throughout twenty-four hours of incubation. Based on these results, we propose that lack (or inhibition) of catabolism is at least as important as efficient anabolic phosphorylation for obtaining therapeutic effects of pyrimidine nucleoside analogs.