The axis IL-10/claudin-10 is implicated in the modulation of aggressiveness of melanoma cells by B-1 lymphocytes.

B-1 lymphocytes are known to increase the metastatic potential of B16F10 melanoma cells via the extracellular signal-regulated kinase (ERK) pathway. Since IL-10 is associated with B-1 cells performance, we hypothesized that IL-10 could be implicated in the progression of melanoma. In the present work, we found that the C57BL/6 mice, inoculated with B16F10 cells that were co-cultivated with B-1 lymphocytes from IL-10 knockout mice, developed fewer metastatic nodules than the ones which were injected with the melanoma cells that were cultivated in the presence of wild-type B-1 cells. The impairment of metastatic potential of the B16F10 cells was correlated with low activation of the ERK signaling pathway, supporting the idea that the production of IL-10 by B-1 cells influences the behavior of the tumor. A microarray analysis of the B-1 lymphocytes revealed that IL-10 deficiency is associated with down-regulation of the genes that code for claudin-10, a protein that is involved in cell-to-cell contact and that has been linked to lung adenocarcinoma. In order to determine the impact of claudin-10 in the cross-talk between B-1 lymphocytes and the B16F10 tumor cells, we took advantage of small interfering RNA. The silencing of claudin-10 gene in B-1 lymphocytes inhibited activation of the ERK pathway and abrogated the B-1-induced aggressive behavior of the B16F10 cells. Thus, our findings suggest that the axis IL-10/claudin-10 is a promising target for the development of therapeutic agents against aggressive melanoma.

Liposomes of phosphatidylcholine and cholesterol induce an M2-like macrophage phenotype reprogrammable to M1 pattern with the involvement of B-1 cells.

Macrophages respond to endogenous and non-self stimuli acquiring the M1 or M2 phenotypes, corresponding to classical or alternative activation, respectively. The role of B-1 cells in the regulation of macrophage polarization through the secretion of interleukin (IL)-10 has been demonstrated. However, the influence of B-1 cells on macrophage phenotype induction by an immunogen that suppress their ability to secrete IL-10 has not been explored. Here, we studied the peritoneal macrophage pattern induced by liposomes comprised of dipalmitoylphosphatidylcholine (DPPC) and cholesterol (Chol) carrying ovalbumin (OVA) (Lp DPPC/OVA), and the involvement of B-1 cells in macrophage polarization. Peritoneal cells from BALB/c, B-1 cells-deficient BALB/xid and C57BL/6 mice immunized with Lp DPPC/OVA and OVA in soluble form (PBS/OVA) were analyzed and stimulated or not in vitro with lipopolysaccharide (LPS). Peritoneal macrophages from BALB/c and C57BL/6 mice immunized with Lp DPPC/OVA showed an M2-like phenotype as evidenced by their high arginase activity without LPS stimulation. Upon stimulation, these macrophages were reprogrammable toward the M1 phenotype with the upregulation of nitric oxide (NO) and a decrease in IL-10 secretion. In addition, high IFN-γ levels were detected in the culture supernatant of peritoneal cells from BALB/c and C57BL/6 mice immunized with Lp DPPC/OVA. Nevertheless, still high levels of arginase activity and undetectable levels of IL-12 were found, indicating that the switch to a classical activation state was not complete. In the peritoneal cells from liposomes-immunized BALB/xid mice, levels of arginase activity, NO, and IL-6 were below those from wild type animals, but the last two products were restored upon adoptive transfer of B-1 cells, together with an increase in IFN-γ secretion. Summarizing, we have demonstrated that Lp DPPC/OVA induce an M2-like pattern in peritoneal macrophages reprogrammable to M1 phenotype after LPS stimulation, with the involvement of B-1 cells.

Cross talk between peritoneal macrophages and B-1 cells in vitro.

B-1 cells constitute a distinct B cell population with unique phenotypic and functional characteristics. They represent the main B cell population found in mouse peritoneal and pleural cavities. The communication between B-1 cells and peritoneal macrophages has been previously studied, and the effect this interaction has on macrophages has been previously described. Using an in vitro co-culture model, herein we demonstrated that peritoneal macrophages were able to increase survival rates and to stimulate proliferation of B-1 cells. IL-6 was also found to be important in B-1 cell survival; recombinant IL-6 increases the percentage of viable B-1 cells in culture. Furthermore, molecules involved in the IL-6 signaling pathway, such as STAT-3 and Bcl-2, were highly expressed in B-1 cells after co-culture with peritoneal macrophages. IL-6-deficient peritoneal macrophages were not able to increase B-1 cell survival, confirming the importance of this cytokine. Altogether, our results indicate a novel mechanism in which peritoneal macrophages are able to regulate the B-1 population via IL-6 secretion.

B16 melanoma cells increase B-1 cell survival, IL-10 production and radioresistance in vitro.

B-1 cells can be differentiated from B-2 cells because they are predominantly located in the peritoneal and pleural cavities and have distinct phenotypic patterns and activation properties. The role of both cell populations in cancer progression is still controversial. Previous studies have indicated that direct contact between B-1 cells and B16 melanoma tumor cells (B16) increases the metastatic potential of the tumor cells. However, cellular changes that are induced in B-1 cells during the interaction between these two cell types have not been evaluated. In the present study, it is hypothesized that B-1 cells are modified after their interaction with tumor cells, leading to both increased cell viability and rate of proliferation. Additionally, soluble factors that were secreted by B16 cells were sufficient to augment B-1 cell viability and to modify the production of IL-10 by B-1 cells. Impressively, after direct or indirect contact with the B16 cells, B-1 cells became resistant to radiation-induced cell death. Thus, future studies that assess the importance of concomitant immunity and other conventional therapies in cancer treatment are needed.

The New Zealand black mouse as a model for the development and progression of chronic lymphocytic leukemia.

BACKGROUND: Similar to a subset of human patients who progress from monoclonal B lymphocytosis (MBL) to chronic lymphocytic leukemia (CLL), New Zealand Black (NZB) mice have an age-associated progression to CLL. The murine disease is linked to a genetic abnormality in microRNA mir-15a/16-1 locus, resulting in decreased mature miR-15a/16. METHODS: Spleens of aging NZB were analyzed for the presence of B-1 cells via flow cytometry and for the presence of a side population (SP) via the ability of cells to exclude Hoechst 33342 dye. The SP was assayed for the presence of hyperdiploid B-1 clones and for the ability to differentiate into B-1 cells in vitro and transfer disease in vivo. In addition, enhanced apoptosis of chemoresistant NZB B-1 cells was examined by restoring miR-16 levels in nutlin-treated cells. RESULTS: Aging NZB mice develop a B-1 expansion and clonal development that evolves from MBL into CLL. An expansion in SP is also seen. Although the SP did contain increased cells with stem cell markers, they lacked malignant B-1 cells and did not transfer disease in vivo. Similar to B-1 cells, splenic NZB SP also has decreased miR-15a/16 when compared with C57Bl/6. Exogenous addition of miR-15a/16 to NZB B-1 cells resulted in increased sensitivity to nutlin. CONCLUSION: NZB serve as an excellent model for studying the development and progression of age-associated CLL. NZB SP cells do not seem to contain cancer stem cells, but rather the B-1 stem cell. NZB B-1 chemoresistance may be related to reduced miR-15a/16 expression.