Isolation of functional mature peritoneal macrophages from healthy humans.

Macrophages play an important role in the inflammatory response. Their various biological functions are induced by different membrane receptors, including Toll-like receptors, which trigger several intracellular signaling cascades and activate the inflammasomes, which in turn elicit the release of inflammatory mediators such as cytokines. In this study, we present a novel method for the isolation of human mature peritoneal macrophages. This method can be easily implemented by gynecologists who routinely perform laparoscopy for sterilization by tubal ligation or surgically intervene in benign gynecological pathologies. Our method confirms that macrophages are the main peritoneal leukocyte subpopulation isolated from the human peritoneum in homeostasis. We showed that primary human peritoneal macrophages present phagocytic and oxidative activities, and respond to activation of the main proinflammatory pathways such as Toll-like receptors and inflammasomes, resulting in the secretion of different proinflammatory cytokines. Therefore, this method provides a useful tool for characterizing primary human macrophages as control cells for studies of molecular inflammatory pathways in steady-state conditions and for comparing them with those obtained from pathologies involving the peritoneal cavity. Furthermore, it will facilitate advances in the screening of anti-inflammatory compounds in the human system.

Expression of LAIR-1 (CD305) on Human Blood Monocytes as a Marker of Hepatic Cirrhosis Progression.

BACKGROUND AND AIM: The presumed role of the inhibitory receptor LAIR-1 (CD305) in the inflammatory response suggests that it might contribute to the pathophysiology of chronic inflammatory diseases such as liver cirrhosis. We studied the LAIR-1 expression on liver macrophages and blood monocytes related to the progression of liver cirrhosis. METHODS: The expression of LAIR-1 was analyzed by immunohistochemistry, flow cytometry, and Western blot. RESULTS: We found a decreased number of macrophages expressing LAIR-1 in cirrhotic liver that could be due to a high presence of collagen, ligand of LAIR-1, in the fibrotic tissue which could downregulate its expression or interfere with the immunostaining. The expression of LAIR-1 decreased after cell differentiation, and the total content, but not the cell surface expression, increased after activation in the HL-60 human macrophage in vitro model. Blood monocytes exhibited higher LAIR-1 expression levels in cirrhotic patients, which were evident even in early clinical stages in all monocyte subsets, and greater in the "intermediate" inflammatory monocyte subpopulation. The in vitro activation of human blood monocytes did not increase its expression on the cell surface suggesting that the in vivo increase of LAIR-1 must be the result of a specific combination of stimuli present in cirrhotic patients. This represents an exclusive feature of liver cirrhosis, since blood monocytes from other chronic inflammatory pathologies showed similar or lower LAIR-1 levels compared with those of healthy controls. CONCLUSIONS: These results may indicate that monocyte LAIR-1 expression is a new biomarker to early detect liver damage caused by chronic inflammation in liver cirrhosis.

Therapeutic potential of pteridine derivatives: A comprehensive review.

Pteridines are aromatic compounds formed by fused pyrazine and pyrimidine rings. Many living organisms synthesize pteridines, where they act as pigments, enzymatic cofactors, or immune system activation molecules. This variety of biological functions has motivated the synthesis of a huge number of pteridine derivatives with the aim of studying their therapeutic potential. This review gathers the state-of-the-art of pteridine derivatives, describing their biological activities and molecular targets. The antitumor activity of pteridine-based compounds is one of the most studied and advanced therapeutic potentials, for which several molecular targets have been identified. Nevertheless, pteridines are also considered as very promising therapeutics for the treatment of chronic inflammation-related diseases. On the other hand, many pteridine derivatives have been tested for antimicrobial activities but, although some of them resulted to be active in preliminary assays, a deeper research is needed in this area. Moreover, pteridines may be of use in the treatment of many other diseases, such as diabetes, osteoporosis, ischemia, or neurodegeneration, among others. Thus, the diversity of the biological activities shown by these compounds highlights the promising therapeutic use of pteridine derivatives. Indeed, methotrexate, pralatrexate, and triamterene are Food and Drug Administration approved pteridines, while many others are currently under study in clinical trials.

Anti-leukemia activity of 4-amino-2-aryl-6,9-dichlorobenzo[g]pteridines.

Pteridines are bicyclic heterocyclic compounds with a pyrazino[2,3-d]pyrimidine nucleus that have shown a wide range of therapeutic utilities. Concretely, 4-aminopteridine derivatives have demonstrated both anti-inflammatory and anti-cancer properties, and some of them, such as methotrexate, are profusely used in medical practice. We have recently synthesized and tested the biological activity of a novel series of 4-amino-2-aryl-6,9-dichlorobenzo[g]pteridines, finding that they present anti-inflammatory properties, as they were able to inhibit in vitro the production of pro-inflammatory cytokines TNF-α and IL-6. Now, we have evaluated the anti-tumor potential of these compounds on HL-60 and K562 leukemia cell lines. Cells growing at exponential rate were exposed to decreasing doses of each compound, from 50 to 0.39 µM, for 24, 48, and 72 h. Cell viability was tested by MTT assay and cell death fashion determined by annexin V/propidium iodide assay. The cytotoxicity of the compounds was determined in differentiated macrophage-like HL-60 cells and in human peripheral blood mononuclear cells to evaluate the potential side effects on quiescent tumor cells and normal cells, respectively. Among the series, compounds 1a, 1b, 1g, 1j, and 1k showed anti-proliferative activity. Compounds 1j and 1k were active against both HL-60 and K562 cells, with a lower IC50 against HL-60 cells. Compounds 1a, 1b, and 1g had a great cytotoxic activity against HL-60, but they were far less potent against K562 cells. None had side effects in differentiated tumor cells or in human peripheral blood mononuclear cells. In conclusion, our results demonstrate that some compounds of this series of 4-amino-2-aryl-6,9-dichlorobenzo[g]pteridines have anti-cancer properties in vitro.

Characterization of human peritoneal monocyte/macrophage subsets in homeostasis: Phenotype, GATA6, phagocytic/oxidative activities and cytokines expression.

Peritoneal macrophages play a critical role in the control of infectious and inflammatory diseases. Although recent progress on murine peritoneal macrophages has revealed multiple aspects on their origin and mechanisms involved in their maintenance in this compartment, little is known on the characteristics of human peritoneal macrophages in homeostasis. Here, we have studied by flow cytometry several features of human peritoneal macrophages obtained from the peritoneal cavity of healthy women. Three peritoneal monocyte/macrophage subsets were established on the basis of CD14/CD16 expression (CD14++CD16-, CD14++CD16+ and CD14highCD16high), and analysis of CD11b, CD11c, CD40, CD62L, CD64, CD80, CD86, CD116, CD119, CD206, HLA-DR and Slan was carried out in each subpopulation. Intracellular expression of GATA6 and cytokines (pro-inflammatory IL-6 and TNF-α, anti-inflammatory IL-10) as well as their phagocytic/oxidative activities were also analyzed, in an attempt to identify genuine resident peritoneal macrophages. Results showed that human peritoneal macrophages are heterogeneous regarding their phenotype, cell complexity and functional abilities. A direct relationship of CD14/CD16 expression, intracellular content of GATA6, and activation/maturation markers like CD206 and HLA-DR, support that the CD14highCD16high subset represents the mature phenotype of steady-state human resident peritoneal macrophages. Furthermore, increased expression of CD14/CD16 is also related to the phagocytic activity.