Figure 4

Vasculotide (VT) suppresses cecal ligation and puncture (CLP)-induced upregulation of proinflammatory cytokines in vivo but does not affect cytokine release from macrophages in vitro. (A) Mice were pretreated with VT (200 ng intraperitoneally) or phosphate-buffered saline (PBS) at 16 hours and 1 hour prior to CLP or sham surgery, respectively. Levels of tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), keratinocyte-derived chemokine/interleukin-8 homologue (KC), macrophage inflammatory protein-2 (MIP-2), and monocyte chemotactic protein-1 (MCP-1) in serum and peritoneal lavage (PL) fluid samples at 18 hours after CLP or sham surgery were quantified by bead-based flow cytometry or enzyme-linked immunosorbent assay (ELISA), respectively. Data are expressed as mean ± standard error of the mean (SEM) (n = 7 to 10 mice per group). *P < 0.05; **P < 0.01 versus sham. †P < 0.05; ‡P < 0.01 versus CLP without VT. (B) Mouse resident peritoneal macrophages were obtained from untreated wild-type mice by PL. Cells were cultured in RPMI 1640/1% fetal calf serum (FCS), preincubated or not with 25 or 100 ng/mL VT for 1 hour, and then stimulated with lipopolysaccharide (LPS) (10 ng/mL) for 4 hours. In some experiments, peritoneal macrophages were preincubated with an sTie-2-Fc chimera (5 μg/mL) 30 minutes prior to the addition of VT. (A) MIP-2 and (B) KC protein levels were analyzed in conditioned medium by specific ELISA. Results are expressed as the mean ± SEM from three independent experiments performed in triplicate. *P < 0.001 versus unstimulated control.