Publications

Results are shown as a dot plot of individual genes, comparing the expression levels found in EV– and SEMA3B-transduced cells. A selected panel of 622 genes, including cytokines and factors mediating inflammatory response (identified by keywords as described in Materials and methods), is highlighted in red. is the only gene in the panel significantly regulated more than twofold. ***, P &lt; 0.001) (B) Real-time quantitative PCR mRNA expression analysis of SEMA3B- and EV-transduced tumor cells using Taqman probes for IL-8 and for the housekeeping gene (internal reference). Reported values indicate fold induction of IL-8 expression in SEMA3B-expressing cells relative to the respective EV control for each cell line, and values correspond to the mean ± SD of three individual experiments. *, P &lt; 0.05. (C) The CM from MDA-MB435 EV control and SEMA3B-expressing cells was collected and concentrated 300 times. 10 μl of this preparation was analyzed by Western blotting with anti–IL-8 antibodies. The indicated amounts of purified IL-8 were loaded for internal reference. The concentration of IL-8 in the original CM of MDA-3B cells was thus estimated to be in the range of 5–7 ng/ml. (D) Western blot analysis of A549 cells expressing SEMA3B under a drug-inducible promoter; control cells (EV) expressed the doxycycline-dependent transactivator rTTA alone. Upon doxycycline treatment, SEMA3B expression in tumor cells (revealed by anti-Myc tag antibodies) and IL-8 levels in the CM (revealed with MAB208) were increased; purified IL8 was loaded as an internal reference. (E) The CM of SEMA3B tumor cells and EV controls were assayed in monocyte migration experiments, similar to those shown in . Where indicated, IL-8 neutralizing antibodies (MAB208) or an unrelated control antibody (i.e., anti-CD19; C Ab), or 100 ng/ml of purified IL-8 were added in the lower well. The number of migrated monocytes was scored as in , and the graph shows the mean of three independent experiments. IL-8 is responsible for the increased monocyte-attracting activity released by SEMA3B-expressing tumor cells. **, P &lt; 0.01.<b>Copyright information:</b>Taken from "The tumor suppressor semaphorin 3B triggers a prometastatic program mediated by interleukin 8 and the tumor microenvironment"The Journal of Experimental Medicine 2008;205(5):1155-1171.Published online 12 May 2008PMCID:PMC2373847.

Abstract Late in erythropoiesis, nuclei are expelled from erythroblasts and 2×1011 anucleated new red blood cells are daily delivered in the peripheral blood. Expelled nuclei expose phosphatidylserine (PS) on their surface, which is used as an “eat me” signal for their engulfment by macrophages located in the blood island. The two PS opsonins, milk-fatglobule EGF8 (MFG-E8) and Growth arrest-specific gene 6 product (GAS6) together with their respective receptors αvβ5/αvβ3 and TAM (TYRO3, AXL and MER), are involved in the phagocytosis of apoptotic cells, but their role in the phagocytosis of expelled nuclei from erythroblasts is not determined. Because fetal liver and bone marrow macrophages do not express MFG-E8, the GAS6-MER pathway might constitute a crucial pathway for the engulfment of nuclei expelled from erythroblasts. To test this hypothesis, we isolated nuclei from late-stage erythroblasts from spleens of phlebotomized mice, and studied nuclei internalization capacity of bone marrow derived macrophages (BMDM) from mice deficient either in GAS6 (GAS6−/−), AXL (AXL−/−) or TYRO3 (TYRO3−/−), or lacking MER kinase domain (MERkd). Released nuclei were identified by flow cytometry according to their size and their double positive staining for the erythroid lineage marker Ter119 and Annexin V for PS. Purity of the preparation was checked by morphological examination of cytospin preparations. In vitro phagocytosis assays show that GAS6−/− BMDM cleared 30% less nuclei than wild-type (WT) BMDM. We observed a slight decrease of internalization capacity for AXL−/− BMDM, whereas TYRO3−/− BMDM engulfed the nuclei as efficiently as WT BMDM. In contrast, MER deficiency nearly abolished nuclei phagocytosis. AXL−/−/TYRO3−/− and AXL−/−/MERkd BMDM were tested and did not show any cumulative effects when compared to WT and single knockouts. We also investigated the signalling pathway downstream of MER in BMDM. In particular, we assessed the expression of the activated form of Rac1, which is crucial for the cytoskeletal reorganization in phagocytosis. Activation of Rac1 after the initiation of the phagocytosis was delayed for 45 minutes in MERkd as compared to WT BMDM. In vivo, we found an accumulation of nuclei in MERkd mice 4 days post phlebotomy, when erythropoiesis is increased in response to anemia. Nuclei circulated in the blood of MERkd mice at a level of 0.08 ± 0.042 G/L and were identified on peripheral blood smears of these mice whereas they were undetectable in the blood of WT mice. We demonstrated an increase of a double labelled Ter119/AnnexinV population corresponding to nuclei in BM (2-fold) and spleen (1.5-fold) of MERkd mice as compared to WT mice. The augmentation of this double labelled population in the MERkd mice translated the phenotype of splenomegaly of these mice. Hematocrit and reticulocyte levels were comparable between WT and MERkd as previously reported (JCI118:583–596, 2008). Thus, MER was critical for in vitro phagocytosis of nuclei from erythroblasts whereas the role of AXL and TYRO3 appeared to be negligible. GAS6 binding to nuclei exposing PS on their surface might form a bridge between PS and MER receptor on BMDM, allowing nuclei clearance. In vivo, the absence of MER caused an accumulation of nuclei in BM and spleen and their appearance in circulating blood due to their inefficient elimination during erythropoietic response to anemia. In conclusion, we postulate that GAS6 and its receptor MER were involved in late erythropoiesis when nuclei are expelled from the erythroblasts and engulfed by BMDM in the blood island, through Rac1 activation.

&lt;p&gt;Supplementary Table 1: Protein quantification of cytokines in tumors and plasma of WT and Flt1TK-/- obese using ELISA; Supplementary Table 2: Quantification of the expression of IL-1ÃŽÃ,² and VEGFR-1 in TAMs from PAN02 tumors by mmunofluorescence.&lt;/p&gt;

Interstitial cells have been implicated in the pathogenesis of renal fibrosis. Given that the urokinase receptor (uPAR) is known to play a role in cell adhesion, migration, and angiogenesis, the present study was designed to evaluate the role of uPAR in the regulation of the phenotypic composition of interstitial cells (macrophages, myofibroblasts, capillaries) in response to chronic renal injury. Groups of uPAR wild-type (+/+) and knockout (-/-) mice were investigated between 3 and 14 d after unilateral ureteral obstruction (UUO) or sham surgery (n = 8 mice per group). The density of F4/80+ interstitial macrophages (Mphi) was significantly lower in the -/- mice (3.3 +/- 0.4 versus 6.9 +/- 1.7% area at day 3 UUO; 10.8 +/- 1.6 versus 15.7 +/- 1.0% at day 14 UUO; -/- versus +/+). In contrast, in the -/- mice there were significantly more alpha smooth muscle actin (alphaSMA)-positive cells (12.9 +/- 3.2 versus 7.8 +/- 1.5% area at day 3 UUO; 21.0 +/- 4.7 versus 9.7 +/- 1.9% at day 14 UUO) and CD34-positive endothelial cells (8.4 +/- 1.9 versus 4.0 +/- 1.1% area at day 14 UUO). These differences were associated with significantly more interstitial fibrosis in the -/- mice based on Sirius red staining (4.6 +/- 0.9 versus 2.3 +/- 0.9% area at 14 d UUO). Absence of the uPAR scavenger receptor was associated with significantly greater accumulation of plasminogen activator inhibitor-1 protein (PAI-1) (20.5 +/- 3.5 versus 9.1 +/- 2.9% area, day 14 UUO) and vitronectin protein (2.4 +/- 1.1 versus 0.9 +/- 0.4% area, day 14 UUO). By immunostaining alphaSMA+ cells, CD34+ cells, vitronectin and PAI-1 co-localized to the same tubulointerstitial area. The number of apoptotic cells increased in response to UUO but was significantly higher in the -/- mice (2.0 +/- 0.2 versus 1.2 +/- 0.2 per 100 tubulointerstitial cells, day 14 UUO) while the number of proliferating cells was significantly lower in the uPAR-/- mice. These data suggest that uPAR deficiency suppresses renal Mphi recruitment, but the absence of this scavenger receptor actually accentuates the fibrogenic response, likely due in part to the delayed clearance of angiogenic/profibrotic molecules such as PAI-1 and decreased receptor-associated uPA activity.

&lt;p&gt;Histopathology pictures of neoplastic abnormalities in Ppp2r4 gene-trapped mice.&lt;/p&gt;