Identification of compounds that decrease numbers of Mycobacteria in human macrophages in the presence of serum amyloid P.

Mϕs are a heterogeneous population of cells and include classically activated Mϕs (M1) and alternatively activated Mϕs (M2). Mϕs can change from M1 to M2 and vice versa in response to environmental stimuli. Serum amyloid P (SAP) is a constitutive plasma protein that polarizes Mϕs to an M2 phenotype, and part of this effect is mediated through FcγRI receptors. In an effort to find ways to alter Mϕs phenotypes, we screened for compounds that can block the SAP-FcγRI interaction. From a screen of 3000 compounds, we found 12 compounds that reduced the ability of fluorescently labeled human SAP to bind cells expressing human FcγRI. Based on cell surface marker expression, 8 of the compounds inhibited the effect of SAP on skewing human Mϕs to an M2 phenotype and in the presence of SAP polarized Mϕs to an M1 phenotype. In diseases, such as tuberculosis, M1s are more effective at killing bacteria than M2s. SAP potentiated the numbers of the mycobacterial strains Mycobacterium smegmatis and Mycobacterium tuberculosis in Mϕs. When added along with SAP, 2 of the compounds reduced intracellular Mycobacterium numbers. Together, these results indicate that the blocking of SAP effects on Mϕs can skew these cells toward an M1 phenotype, and this may be useful in treating diseases, such as tuberculosis.

Improved serum-free culture conditions for the differentiation of human and murine fibrocytes.

CD14+ peripheral blood monocytes can differentiate into fibroblast-like cells called fibrocytes. Fibrocytes are associated with, and are at least partially responsible for, wound healing and fibrosis in multiple organ systems. In a variety of lesions in vivo, monocytes appear to differentiate into fibrocytes within days. However, in vitro culture conditions can take up to two weeks to generate fibrocytes. In this study, we describe enhanced serum-free conditions that support the rapid differentiation of human and murine fibrocytes. We compared the effect on fibrocyte differentiation of different anti-coagulants used when collecting blood, and for culturing cells, the effects of different commercial media formulations, the addition of a variety of supplements, cell density, conditioned medium, and glass and plastic substrates. We found that both heparin and EDTA were suitable anti-coagulants, but that blood treated with citrate-phosphate dextrose led to a reduced number of fibrocytes. Fibrocyte differentiation was enhanced when the serum-free medium was based on either FibroLife or StemPro formulations. We also found that only positively charged or hydrophilic glass and plastic surfaces provide adequate support for fibrocyte differentiation. Finally, the optimal cell density was 2.5 x 10(5)cells/ml (approximately 800 cells per mm(2)). These results indicate that blood collection, substrates, media, and cell density all influence in vitro fibrocyte differentiation.

Cells respond to and bind countin, a component of a multisubunit cell number counting factor.

In Dictyostelium discoideum counting factor (CF), a secreted approximately 450-kDa complex of polypeptides, inhibits group and fruiting body size. When the gene encoding countin (a component of CF) was disrupted, cells formed large groups. We find that recombinant countin causes developing cells to form small groups, with an EC(50) of approximately 3 ng/ml, and affects cAMP signal transduction in the same manner as semipurified CF. Recombinant countin increases cell motility, decreases cell-cell adhesion, and regulates gene expression in a manner similar to the effect of CF. However, countin does not decrease adhesion or group size to the extent that semipurified CF does. A 1-min exposure of developing cells to countin causes an increase in F-actin polymerization and myosin phosphorylation and a decrease in myosin polymerization, suggesting that countin activates a rapid signal transduction pathway. (125)I-Labeled countin has countin bioactivity, and binding experiments suggest that vegetative and developing cells have approximately 53 cell-surface sites that bind countin with a K(D) of approximately 1.5 ng/ml or 60 pm. We hypothesize that countin regulates cell development through the same pathway as CF and that other proteins within the complex may modify the activity of countin and/or have independent size-regulating activities.