TLR2/6-dependent stimulation by MALP-2 promotes migration and GM-CSF release of leukocytes. (A) Migration of human leukocytes (1 × 105) was carried out in transwell cell-culture inserts for 24 hours after stimulation with different concentrations of MALP-2 as indicated; 10% FCS was used as positive control (n = 8). *P < .05, **P < .01 vs control. (B) Migration of human leukocytes (1 × 105) was carried out in transwell cell-culture inserts for 24 hours. As chemoattractant, supernatants from leukocytes after 24 hours stimulation with MALP-2 (1 μg/mL) were used and compared with control supernatant from unstimulated leukocytes (n = 6). *P < .05 vs control. (C) GM-CSF levels in supernatants of 1.5 × 104 and 1.5 × 105 human leukocytes after stimulation with MALP-2 (1 μg/mL) or with different concentrations of MALP-2 for 24 hours as indicated determined by ELISA (n = 7). *P < .05, **P < .01 vs control. (D) GM-CSF release from HUVECs (1.5 × 104) and human leukocytes (1.5 × 104) after stimulation with MALP-2 (1 μg/mL) for 24 hours assessed by ELISPOT. Representative pictures of 3 independent experiments are shown. (E) GM-CSF levels in sorted lymphocytes, granulocytes, and monocytes (1.5 × 105) after stimulation with MALP-2 (1 μg/mL) for 24 hours as determined by ELISA (n = 5). *P < .05 vs control. (F) Immunofluorescent staining of Matrigel plaques after the administration of MALP-2 (1 μg/mL) for 6 days with antibodies against CD31 and MOMA-2. Representative pictures of 3 independent experiments are shown (Leica DM 4000B microscope, 20×/0.50 NA dry objective, Leica DFC 320 camera, Leica QWin Version 3 software). (A-C,E) Error bars represent mean ± SEM.
