Background: Upon stimulation with different cytokines, macrophages can undergo classical or alternative activation to become M1 or M2 macrophages. Alternatively activated (or M2) macrophages are defined by their expression of specific gene products and play an important role in containing inflammation, removing apoptotic cells and repairing tissue damage. Whereas it is well-established that IL-4 can drive alternative activation, if lack of TGFβ signaling at physiological levels affects M2 polarization has not been addressed.Results: Vav1-Cre x TβRII^fx/fxmice, lacking TβRII function in hematopoietic cells, exhibited uncontrolled pulmonary inflammation and developed a lethal autoimmune syndrome at young age. This was accompanied by significantly increased numbers of splenic neutrophils and T cells as well as elevated hepatic macrophage infiltration and bone marrow monocyte counts.TβRII^-/-CD4⁺and CD8⁺T-cells in the lymph nodes and spleen expressed increased cell surface CD44, and CD69 was also higher on CD4⁺lymph node T-cells. Loss of TβRII in bone marrow-derived macrophages (BMDMs) did not affect the ability of these cells to perform efferocytosis. However, these cells were defective in basal and IL-4-inducedarg1mRNA and Arginase-1 protein production. Moreover, the transcription of genes that are typically upregulated in M2-polarized macrophages, such asym1, mcr2 and mgl2,was also decreased in peritoneal macrophages and IL-4-stimulatedTβRII^-/-BMDMs. We found that cell surface and mRNA expression of Galectin-3, which also regulates M2 macrophage polarization, was lower inTβRII^-/-BMDMs. Very interestingly, the impaired ability of these null mutant BMDMs to differentiate into IL-4 polarized macrophages was Stat6- and Smad3-independent, but correlated with reduced levels of phospho-Akt and β-catenin.Conclusions: Our results establish a novel biological role for TGFβ signaling in controlling expression of genes characteristic for alternatively activated macrophages. We speculate that lack of TβRII signaling reduces the anti-inflammatory M2 phenotype of macrophages because of reduced expression of these products. This would cause defects in the ability of the M2 macrophages to negatively regulate other immune cells such as T-cells in the lung, possibly explaining the systemic inflammation observed inVav1-Cre x TβRII^fx/fxmice.

The Fer protein belongs to the fes/fps family of nontransmembrane receptor tyrosine kinases. Lack of success in attempts to establish a permanent cell line overexpressing it at significant levels suggested a strong negative selection against too much Fer protein and pointed to a critical cellular function for Fer. Using a tetracycline-regulatable expression system, overexpression of Fer in embryonic fibroblasts was shown to evoke a massive rounding up, and the subsequent detachment of the cells from the substratum, which eventually led to cell death. Induction of Fer expression coincided with increased complex formation between Fer and the cadherin/src-associated substrate p120^cas and elevated tyrosine phosphorylation of p120^cas. β-Catenin also exhibited clearly increased phosphotyrosine levels, and Fer and β-catenin were found to be in complex. Significantly, although the levels of α-catenin, β-catenin, and E-cadherin were unaffected by Fer overexpression, decreased amounts of α-catenin and β-catenin were coimmunoprecipitated with E-cadherin, demonstrating a dissolution of adherens junction complexes. A concomitant decrease in levels of phosphotyrosine in the focal adhesion-associated protein p130 was also observed. Together, these results provide a mechanism for explaining the phenotype of cells overexpressing Fer and indicate that the Fer tyrosine kinase has a function in the regulation of cell-cell adhesion.

B-cell precursor acute lymphoblastic leukemia and stromal cells communicate through Galectin-3

The bone marrow (BM) stromal niche can protect acute lymphoblastic leukemia (ALL) cells against the cytotoxicity of chemotherapeutic agents and is a possible source of relapse. The stromal-derived factor-1 (SDF-1)/CXCR4 axis is a major determinant in the crosstalk between leukemic cells and BM stroma. In this study, we investigated the use of AMD11070, an orally available, small-molecule antagonist of CXCR4, as an ALL-sensitizing agent. This compound effectively blocked stromal-induced migration of human ALL cells in culture and disrupted pre-established adhesion to stroma. To examine how to optimally use this compound in vivo, several combinations with cytotoxic drugs were tested in a stromal co-culture system. The best treatment regimen was then tested in vivo. Mice transplanted with murine Bcr/Abl ALL cells survived significantly longer when treated with a combination of nilotinib and AMD11070. Similarly, immunocompromised mice transplanted with human ALL cells and treated with vincristine and AMD11070 had few circulating leukemic cells, normal spleens and reduced human CD19+ cells in the BM at the termination of the experiment. These results show that combined treatment with AMD11070 may be of significant benefit in eradicating residual leukemia cells at locations where they would otherwise be protected by stroma.

Small GTPases of the Rho family are key regulators of phagocytic leukocyte function. Abr and Bcr are homologous, multidomain proteins. Their C-terminal domain has GTPase-activating protein (GAP) activity that, in vitro, is specific for Rac and Cdc42. To address the in vivo relevance of these entire proteins, of which little is known, the current study examined the effect of the genetic ablation of Abr and Bcr in murine macrophages. The concomitant loss of Abr and Bcr induced multiple alterations of macrophage cellular behavior known to be under the control of Rac. Macrophages lacking both Abr and Bcr exhibited an atypical, elongated morphology that was reproduced by the ectopic expression of GAP domain mutant Abr and Bcr in a macrophage cell line and of constitutively active Rac in primary macrophages. A robust increase in colony-stimulating factor 1 (CSF-1)-directed motility was observed in macrophages deficient for both proteins and, in response to CSF-1 stimulation, Abr and Bcr transiently translocated to the plasma membrane. Phagocytosis of opsonized particles was also increased in macrophages lacking both proteins and correlated with sustained Rac activation. Bcr and Abr GAP mutant proteins localized around phagosomes and induced distinct phagocytic cup formation. These results identify Abr and Bcr as the only GAPs to date that specifically negatively regulate Rac function in vivo in primary macrophages.