The adaptor protein SLy2 (Src homology domain 3 lymphocyte protein 2) is located on human chromosome 21 and was reported to be among a group of genes amplified in Down's syndrome (DS) patients. DS patients characteristically show an impaired immunity to pneumococcal infections. However, molecular mechanisms linking gene amplifications with specific DS phenotypes remain elusive. To investigate the effect of SLy2 gene amplification on the mammalian immune system, we studied SLy2 overexpressing transgenic-SLy2 (TG) mice. We found that baseline immunoglobulin M (IgM) levels as well as IgM responses following Pneumovax immunizations were reduced in TG mice. Moreover, B-1 cells, the major natural IgM-producing population in mice, were reduced in the peritoneal cavity of TG mice, while other immune cell compartments were unaltered. Mechanistically, SLy2 overexpression attenuated the expression of the IL-5 receptor α chain on B-1 cells, resulting in decreased B-1 cell numbers and decreased differentiation into Ab-secreting cells. Since B-1 cells essentially contribute to immunity against Streptococcus pneumoniae, the present study provides a novel molecular link between SLy2 expression and pneumococcal-specific IgM responses in vivo. These studies suggest that the adaptor protein SLy2 is a potential future target for immunomodulatory strategies for pneumococcal infections.

Fluorescently labeled Ly6G antibodies enable the tracking of neutrophils in mice, whereas purified anti-Ly6G rapidly depletes neutrophils from the circulation. The mechanisms underlying neutrophil depletion are still under debate. Here, we examined how identical Ly6G antibodies coupled to different fluorochromes affect neutrophil fate in vivo. BM cells stained with Ly6G antibodies were injected into mice. The number of retrieved anti-Ly6G-FITC(+) cells was reduced significantly in comparison with anti-Ly6G-APC(+) or anti-Ly6G-PE(+) cells. Flow cytometry and multispectral imaging flow cytometry analyses revealed that anti-Ly6G-FITC(+) neutrophils were preferentially phagocytosed by BMMs in vitro and by splenic, hepatic, and BM macrophages in vivo. Direct antibody injection of anti-Ly6G-FITC but not anti-Ly6G-PE depleted neutrophils to the same degree as purified anti-Ly6G, indicating that the FITC-coupled antibody eliminates neutrophils by a similar mechanism as the uncoupled antibody. With the use of a protein G-binding assay, we demonstrated that APC and PE but not FITC coupling inhibited access to interaction sites on the anti-Ly6G antibody. We conclude the following: 1) that neutrophil phagocytosis by macrophages is a central mechanism in anti-Ly6G-induced neutrophil depletion and 2) that fluorochrome-coupling can affect functional properties of anti-Ly6G antibodies, thereby modifying macrophage uptake of Ly6G-labeled neutrophils and neutrophil retrieval following adoptive cell transfer or injection of fluorescent anti-Ly6G.

Estrogen receptor-alpha (ERα) is a key factor in the development of breast cancer in humans. The expression and activity of ERα is regulated by a multitude of intracellular and extracellular signals. Here we show a cross-talk between β-catenin and ERα in human breast cancer cells. Knockdown of β-catenin by RNAi resulted in significant reduction of ERα mRNA and/or protein levels in MCF-7, T-47D, and BT-474 breast cancer cells and in significant reduction of estradiol-induced expression of the ERα target genes pS2 and GREB1. In addition β-catenin silencing resulted in significant decrease of growth of MCF-7 cells both in the absence and presence of estradiol. β-catenin and ERα could not be co-immunoprecipitated by ERα antibodies from lysates of E2-treated or untreated cells suggesting lack of direct physical interaction. It is concluded that β-catenin is a positive regulator of ERα mRNA and protein expression.