Figure 1.
Delayed skin wound healing in Fpr double deficient Fpr1/2-/-mice.
A, representative pictures of skin wound of WT and Fpr1/2-/- mice. A 6-mm full-thickness (including the Panniluluscarnosus) skin was excised from the right and left upper paravertebral regions of each animal and the injured areas were measured daily using NIH Image J software (version 1.37) (n = 5). B, the areas of wounds. Mice were punched to generate two 6-mm full thickness skin wounds with or without IP injection CRAMP (10 µg/100 µl) or Boc-2 (5 µM, 100 µl) (n = 15). The areas of wound were calculated. *, significantly increased wound area in Fpr1/2-/-and Boc-2 treated mice, compared with the wound area of WT mice at the same time points (p< 0.05).
Figure 2.
Reduced neutrophil infiltration in the wounds of Fpr-deficient mice and the production of chemokines.
A, representative immunofluorescence of skin wound showing Ly6G+ cells 4 h after injury. Cryosections of wounded skin from WT and Fpr-deficient mice were labeled with Ly6G and DAPI (Red: Ly6G; Blue: DAPI) (n = 5, scale bar: 20 µm). Insert: control IgG staining. B, Kinetics of infiltrating Ly6G+ neutrophils in 3 consecutive high power fields (HPF). *, significantly reduced Ly6G+ cells in the wounds of Fpr-deficient micecompared with WT mice (p< 0.05). #, significantly reduced Ly6G+ cells in the wounds of Boc-2 pretreated WT mice, compared with WT mice without pretreatment (p< 0.05). C&D, chemokinesCXCL1 and CXCL2 in the homogenates of skin wound from WT and Fpr-deficient mice. Skin (1 cm×1 cm) with wounds in the center was excised and homogenized in 5 ml DPBS. The homogenates were centrifuged and the supernatants were collected for measurement of CXCL1 and CXCL2 by ELISA (n = 15).
Figure 3.
Chemokine production and chemotactic activity of homogenates of skin wound.
A-C, chemokine production of skin wound at 72 h (n = 15). WT and Fpr-deficient mice were subjected to full-thickness skin wound and the wounds were harvested at 72 h after injury and then homogenized for chemokine measurement with ELISA. D, homogenate-induced migration of parental and Fpr-transfected HEK293 cells. Migrating cells in response to different concentration of the homogenate in 3 HPF were counted. *, significantly increased migrating cells in response to homogenates as compared to medium control (0) (p<0.05). E, chemotactic activity of skin homogenates for neutrophils from WT and Fpr-deficient mice. *, significantly increased migrating neutrophils in response to homogenates as compared to medium control (0) (p<0.05).
Figure 4.
Fpr agonist activity in the skin wound.
A - C, inhibition of homogenate-induced migration of neutrophils from WT mice by Fpr antagonists (n = 5). Neutrophils from WT mice were pretreated with Boc-1 (4 µM and 8 µM, A), WRW4 (4 µM and 8 µM, B) or Boc-2 (5 µM and 10 µM, C) for 30 min and then were measured for chemotaxis in response to homogenates of wounded skin collected at 4 h. *, significantly decreased number of migrating cells treated with Fpr antagonists as compared to neutrophils without pretreatment (p< 0.05). #, significantly decreased number of migrating cells treated with Fpr antagonists as compared to neutrophils without pretreatment (p< 0.05). D, inhibition of the chemotactic activity of skin wound homogenates by a neutralizing CRAMP antibody. HEK293/Fpr2 cells were measured for chemotaxis in response to CRAMP or skin wound homogenates with or without pretreatment by a neutralizing CRAMP antibody (10 µg/ml for 45 min) (n = 5). *, significantly increased number of migrating cells in response to CRAMP or homogenates as compared to medium control (0) (p< 0.05). #, significantly decreased number of migrating cells as compared to neutrophils without pretreatment by CRAMP antibody (p< 0.05).