toward End Target Chemoattractants Fer Kinase Limits Neutrophil Chemotaxis

Neutrophil recruitment and directional movement toward chemotactic stimuli are important processes in innate immune responses. This study examines the role of Fer kinase in neutrophil recruitment and chemotaxis to various chemoattractants in vitro and in vivo. Mice targeted with a kinase-inactivating mutation (Fer DR/DR ) or wild type (WT) were studied using time-lapse intravital microscopy to examine leukocyte recruitment and chemotaxis in vivo. In response to keratinocyte-derived cytokine, no difference in leukocyte chemotaxis was observed between WT and Fer DR/DR mice. However, in response to the chemotactic peptide WKYMVm, a selective agonist of the formyl peptide receptor, a 2-fold increase in leukocyte emigration was noted in Fer DR/DR mice ( p < 0.05). To determine whether these defects were due to Fer signaling in the endothelium or other nonhematopoietic cells, bone marrow chimeras were generated. WKYMVm-induced leukocyte recruitment in chimeric mice (WT bone marrow to Fer DR/DR recipients or vice versa) was similar to WT mice, suggesting that Fer kinase signaling in both leukocytes and endothelial cells serves to limit chemotaxis. Puriﬁed Fer DR/DR neutrophils demonstrated enhanced chemotaxis toward end target chemoattractants (WKYMVm and C5a) compared with WTusing an under-agarose gel chemotaxis assay. These defects were not observed in response to intermediate chemoattractants (keratinocyte-derived cytokine, MIP-2, or LTB 4 ). Increased WKYMVm-induced chemotaxis of Fer DR/DR neutrophils correlated with sustained PI3K activity and reduced reliance on the data in 21). These studies suggest that Fer plays a role in limiting leukocyte recruitment across endothelial and epithelial barriers in response to bacterial Ag. In this study, we demonstrate, in vitro and in vivo, that Fer plays a role in leukocyte chemotaxis toward the fMLP-like peptide, WKYMVm, but not toward keratinocyte-derived cytokine (KC). Bone marrow chimeras were used to determine the role of leukocyte and endothelial Fer in WKYMVm-induced chemotaxis. Furthermore, using puriﬁed neutrophils from wild-type (WT) and Fer DR/DR mice, we observed a role for Fer in limiting neutrophil chemotaxis toward WKYMVm or C5a, but not to intermediate chemoattractants. This correlated with skewed signaling in Fer-deﬁcient neutrophils away from p38 MAPK-based chemotaxis to PI3K-driven chemotaxis. Our data suggest that Fer kinase is a stimulus-speciﬁc inhibitory kinase for leukocyte recruitment in vivo and for neutrophil chemotaxis in vitro by modulating PI3K activity. gel HBSS:dH 2 O A m l aliquot of this so- lution the chemoattractant was added to this aliquot to the desired ﬁnal concentration. A small amount of india ink was added to the gel to enable visualization on the cremaster muscle, and the gel was held in place with a cover slip. Single unbranched venules (25–40 m m in diameter) were selected for study, and intravascular leukocyte kinetics was observed in the same section of venule in 5-min periods at intervals (zero; before gel placement, 30- and 60-min postgel placement) during the experiment. The number of rolling and adherent leukocytes was determined off-line during video playback analysis. Rolling leukocytes were deﬁned as those cells moving at a velocity less than that of erythrocytes within a given vessel. Leukocyte rolling velocity was determined by measuring the time required for a leu- kocyte to roll along a 100- m m length of venule. Rolling velocity was determined for 20 leukocytes at each time interval. Leukocytes were considered adherent to the venular endothelium if they remained stationary or Leukocyte as the number of extravascular leukocytes in the microscopic ﬁeld of between the vessel and the gel (a region of ∼ 200 3 300 mm). Venular diameter was measured on-line using a video caliper (Microcirculation Research Institute, A&M College Station, Centerline RBC ve- locity (V RBC ) was also measured on-line using an optical Doppler velo-cimeter (Microcirculation Research Institute), and mean RBC velocity was determined as V RBC Extravascular leukocyte chemotaxis was time-lapse recorded over 1 h. Leukocyte chemotaxis was determined in WT or Fer DR/DR mice or in bone marrow chimeric mice described below. both WTand Fer DR/DR neutrophils. The optimal dose of WKYMVm for neutrophil chemotaxis in vitro was observed at 1 m M in both groups. Signiﬁcantly more Fer DR/DR neutrophils moved toward WKYMVm at all concentrations relative to the WT cells. These data demonstrate enhanced leukocyte chemotaxis in vitro, demonstrating the importance of Fer kinase within the leukocyte as a negative regulator of chemotaxis.