NEUTRALIZATION OF KC AND MIP-2; DIVERGENT EFFECTS ON ACTIVATION PATHWAYS IN MOUSE PMN CELL LINE -CONSISTENT WITH MOUSE BLOOD PMN

Abstract

Acute lung injury (ALI) is associated with the targeting and sequestration of PMN in the lung. Chemokines, produced by resident and non-resident pulmonary cells in response to inflammatory stimuli, activate pathways that regulate PMN chemotaxis, apoptosis and cytolytic activity. We have shown that systemic antibody blockade of murine PMN chemokines keratinocyte-derived chemokine (KC) and macrophage inflammatory protein-2 (MIP-2) and local pulmonary silencing with KC or MIP-2 siRNA differentially effect lung tissue injury and inflammation in a mouse model of hemorrhage (Hem) induced PMN priming for ALI following septic challenge. To further investigate the divergent roles KC and MIP-2 play while sharing affinity for a common CXCR2-like receptor, we examined phosphorrylation patterns for proteins associated with PMN activation. Previously we reported differences in the %-phospho-AKT, ERK1/2 and p38, 12-14H-post systemic antibody neutralization of KC or MIP-2 following Hem. This data suggested that KC and MIP-2 function through different downstream signaling pathways. In order to further test our hypothesis of divergent activation pathways, an inducible, terminally differentiated PMN cell line (MPRO) was used. MPRO cells, 3 × 104 cells/well in 96 well plates, were stimulated with 50 nM platelet activating factor and treated with either 100 ng/ml anti-KC or 25 ng/ml anti-MIP-2 or IgG. Phospho- vs. total protein was assessed for pathway activation proteins AKT, ERK1/2 and p38 by ELISA method (SuperArray CASE Kit). Phosphorylation profiles for antibody vs. IgG treated MPRO cells for AKT, ERK1/2 and p38 and were consistent with those obtained from mouse blood PMN. The %-phospho-p38 and AKT decreased below IgG levels in the anti-MIP-2 treated cells. While %-phospho-ERK1/2 was decreased below IgG, there was no difference between antibody treatments. These findings support our hypothesis that murine PMN chemokines, KC and MIP-2, differentially stimulate activation pathways that regulate PMN activity. (NIH HL73525)