The short chain fatty acid sodium butyrate regulates the induction of CD1a in developing dendritic cells.

Dendritic cells (DCs) are professional antigen-presenting cells with attributes for priming/activating T cells and mediating immune responses. Considering the importance of DCs in the initiation of immune responses, it will be of interest to study their mechanisms of regulation. Histone-modifying enzymes, such as histone deacetylases (HDACs), are critical in controlling chromatin organization. The aim of our study was to investigate DC differentiation under the influence of sodium butyrate (NaB), a short chain fatty acid that is a histone deacetylase inhibitor. Monocytes from healthy individuals were differentiated into immature DCs with IL-4 and GM-CSF in the presence or absence of NaB. DC differentiation was evaluated by CD14 and CD1a expression by flow cytometry. We observed that monocytes stimulated to differentiate in the presence of NaB displayed colony formation and dendritic cell morphology, lost CD14 and showed decreased secretion of IL-1ß. The acquisition of CD1a, however, was impaired. Being a natural short chain fatty acid, NaB may regulate CD1a acquisition independently of its HDAC inhibitory activity. We observed that the addition of peroxisome proliferator-activated receptor γ (PPAR-γ) antagonist (GW9662) did not reverse NaB effect, suggesting this was not the pathway involved. On the other hand, CD1a can also be induced by toll like receptors 2 (TLR 2) agonists, such as Pam3Cys, and NaB inhibited this effect. Our data suggest that the histone deacetylase inhibitor NaB instead of impairing DC differentiation inhibits the acquisition of CD1a induced both by cytokines and by TLR 2 agonist stimulus. Furthermore, this occurs at the transcriptional level as NaB led to a decrease in mRNA levels of CD1a and upregulation of CD1d.

Simultaneous tissue factor expression and phosphatidylserine exposure account for the highly procoagulant pattern of melanoma cell lines.

A correlation between cancer and hypercoagulability has been described for more than a century. Patients with cancer are at increased risk for thrombotic complications, and the clotting initiator protein, tissue factor (TF), is possibly involved in this process. In addition to TF, the presence of negatively charged phospholipids, particularly phosphatidylserine (PS), is necessary to support some of the blood-clotting reactions. There are few reports describing PS exposure by tumor cells. In this study, we characterized the procoagulant properties of the murine B16F10 and the human WM-266-4 melanoma cell lines. Flow cytometry analyses showed constitutive TF expression by both cell lines, in contrast to negative staining observed for the nontumorigenic melanocyte lineage, melan-A. In addition, tumor cells accelerate plasma clotting in a number-dependent manner. For WM-266-4, this ability was partially reversed by an anti-TF antibody but not by aprotinin, a nonspecific serine-protease inhibitor. Furthermore, flow-cytometric analyses showed the presence of PS at the outer leaflet of both cell lines. This phenomenon was determinant for the assembly of the intrinsic tenase (FIXa/FVIIIa) and prothrombinase (FXa/FVa) complexes, resulting in the activation of FX to FXa and prothrombin to thrombin, respectively. As a result, incubation of WM-266-4 with human plasma produces robust thrombin generation. In conclusion, simultaneous TF expression and PS exposure are responsible for the highly procoagulant pattern of the aggressive melanoma cell lines B16F10 and WM-266-4. Therefore, these cell lines might be regarded as useful models for studying the role of blood coagulation proteins in tumor biology.

Modulation of the immune system by ouabain.

Ouabain, a known inhibitor of the Na,K-ATPase, has been shown to regulate a number of lymphocyte functions in vitro and in vivo. Lymphocyte proliferation, apoptosis, cytokine production, and monocyte function are all affected by ouabain. The ouabain-binding site occurs at the alpha subunit of the enzyme. The alpha subunit plays a critical role in the transport process, and four different alpha-subunit isoforms have been described with different sensitivities to ouabain. Analysis by RT-PCR indicates that alpha1, alpha2, and alpha3 isoforms are all present in murine lymphoid cells obtained from thymus, lymph nodes, and spleen. In these cells ouabain exerts an effect at concentrations that do not induce plasma membrane depolarization, suggesting a mechanism independent of the classical inhibition of the pump. In other systems, the Na,K-ATPase acts as a signal transducer in addition to being an ion pump, and ouabain is capable of inducing the activation of various signal transduction cascades. Neither resting nor concanavalin A (Con A)-activated thymocytes had their levels of phosphorylated-extracellular signal-regulated kinase (P-ERK) modified by ouabain. However, ouabain decreased p38 phosphorylation induced by Con A in these cells. The pathway induced by ouabain in lymphoid cells is still unclear but might vary with the type and state of activation of the cell.