Involvement of placental G-CSF in the in vitro secretion of sFlt-1 by peritoneal macrophages

LITWIN S, PRADOS MB, CORTINA ME, ROUX ME, MIRANDA S

Berlin, Alemania

Congreso; 5th EUROPEAN CONGRESS OF REPRODUCTIVE IMMUNOLOGY; 2007

We previously reported that multiparous placentae showed an increase in the number of macrophages and in the invasive trophoblast cells, which highly expressed G-CSF, M-CSF, VEGF and its receptor Flt-1. Here we investigated whether G-CSF and M-CSF were able to modify the secretion of VEGF and sFlt-1 by isolated macrophages. M&M: term placentae were obtained from normal CBA/JxBALB/c and abortion prone CBA/JxDBA/2 crossbreedings and divided into 3 groups: primiparous young (PY: 3 months old), primiparous old (PO: 8,5 months old) and multiparous old (MO: 87ml,5 months old and 4 pregnancies). Placentae were minced and cultured (76 ± 1 mg/ml). G-CSF and M-CSF were checked in the placental supernatants (PS) by western blot. Both factors were neutralized or not in the PS with specific antibodies. Non inflammatory peritoneal macrophages were obtained from BALB/c mice, cultured (3x106 ml) alone (CM) or in the presence of 10% of the different neutralized (ReN) and not neutralized (Re) PS. Controls of PS were also carried out (CPS). Besides, cells were cultured with rmG-CSF or rmM-CSF (respectively 500-250-125-75 ng/ml) or with 125 ng/ml of different mixtures (80/20; 60/40; 40/60; 20/80). VEGF ans sFlt-1 were investigated by ELISA. Results: VEGF (pg/ml): CM: 35±15; CPS: 80±20; Re and ReN: not detected (nd). sFlt-1 (ng/ml): CM: nd; CPS: 36-47; Re: 140±30; ReN with anti-G-CSF diminishing of approx. %50; ReN with anti-M-CSF mild inhibition. The addition of rm-G-CSF or rmM-CSF or the mixtures did not induce secretion of sFlt-1. Our results might indicate that G-CSF in combination with other/s factor/s secreted by placentae induces the in vitro secretion of sFlt-1 by macrophages secuestrating the VEGF produced. We suggest that this mechanism may partially explain the increase of Flt-1 observed in multiparous placentae.