Or-dependent migration toward chemokine gradient is essential. A recent study found that CX3CR1 deficiency resulted in Indolactam V chemical information decreased recruitment of CX3CR1-positive myeloid cells into the burn wound leading to decreased wound healing [19]. Another study found that CCR2 was important for neutrophil tissue infiltration during sepsis [20]. This chemokine receptor pathway may be an attractive therapeutic approach for wound healing [21], however the specific role played by CCR2 on macrophage in severe burn and sepsis is yet to be examined. Taking into consideration that the effects of NE on macrophage function are not fully understood, the present study investigates the effects of NE on macrophage proliferation, maturation and function in a murine bone marrow ex vivo culturing system. We found that NE has a broad regulating effect on macrophage differentiation, maturation and activities such as phagocytosis, migration and cytokine secretion. Our findings provide new insights into the mechanisms by which the catecholamines modulate the immune response in severely traumatized patients.CA, USA). MafB polyclonal primary antibody and K162 web FITCconjugated goat anti-rabbit IgG were from Abcam Inc. (Toronto, ON, Canada).Animal and bone marrow-derived macrophage cultureAll of the female C57BL/6 mice were purchased from Charles River Laboratories (St Constant, Quebec, Canada). All animals were housed maximum five per cage and maintained on a constant light: dark, 12:12 cycle. All animal procedures were approved by the Sunnybrook Health Science Center Animal Care Committee. Murine bone marrow-derived macrophages were generated as previously described [22]. Bone marrow cells were prepared from femur and tibial bone marrow of C57BL/6 mice and the concentration was adjusted at 2 x 106/mL. BMM cells were cultured in RPMI 1640 lacking phenol red supplemented with 40 ng/mL of mouse M-CSF, 2 mM glutamine, 100 U penicillin/ 0.1mg streptomycin/mL, 10 mM HEPES buffer, 10 of charcoal-dextran-treated FBS instead of regular FCS. Total BM cells were seeded at 1 x 106 cells per mL in 2 mL of media in a 24-well plate and treated with or without NE. Various doses of NE (final concentrations, 1 x 10-8 M or 1 x 10-6 M) were added at day 0. On day 3 and day 6, half of the culture media were removed and replaced with fresh hormonedeficient media containing 40 ng/mL of M-CSF. Macrophages were harvested on day 7. To activate macrophage, 50 ng/mL of LPS was added to the culture media at day 6 and cultured for another 24 hours. For the time point study, in addition to day 0, NE (final concentration, 1 x 10-6 M) was added on day 3 and day 6. To evaluate the effects of NE on macrophage number, cells were counted immediately upon the completion of the cell culturing.Cell proliferation assayBMM proliferation regulated by NE was examined using the CyQUANT Cell Proliferation Assay Kit according to manufacturers instructions. Briefly, bone marrow cells were isolated as mentioned above and 2 x 105 BM cells were seeded into each well of a 96-well plate and treated with various doses of NE (final concentrations, 1 x 10-6 M and 1 x 10-8 M) or without NE. On day 7, media were 23977191 carefully removed, washed with PBS and plates were placed in a -80 freezer. The standard curve was made using BMMs harvested from culture flask in accordance with the protocol. Based on the standard curve, cell numbers in each sample can be calculated.Materials and MethodsReagents and AbsFITC-Dextran (MW: 40K) and LPS from Es.Or-dependent migration toward chemokine gradient is essential. A recent study found that CX3CR1 deficiency resulted in decreased recruitment of CX3CR1-positive myeloid cells into the burn wound leading to decreased wound healing [19]. Another study found that CCR2 was important for neutrophil tissue infiltration during sepsis [20]. This chemokine receptor pathway may be an attractive therapeutic approach for wound healing [21], however the specific role played by CCR2 on macrophage in severe burn and sepsis is yet to be examined. Taking into consideration that the effects of NE on macrophage function are not fully understood, the present study investigates the effects of NE on macrophage proliferation, maturation and function in a murine bone marrow ex vivo culturing system. We found that NE has a broad regulating effect on macrophage differentiation, maturation and activities such as phagocytosis, migration and cytokine secretion. Our findings provide new insights into the mechanisms by which the catecholamines modulate the immune response in severely traumatized patients.CA, USA). MafB polyclonal primary antibody and FITCconjugated goat anti-rabbit IgG were from Abcam Inc. (Toronto, ON, Canada).Animal and bone marrow-derived macrophage cultureAll of the female C57BL/6 mice were purchased from Charles River Laboratories (St Constant, Quebec, Canada). All animals were housed maximum five per cage and maintained on a constant light: dark, 12:12 cycle. All animal procedures were approved by the Sunnybrook Health Science Center Animal Care Committee. Murine bone marrow-derived macrophages were generated as previously described [22]. Bone marrow cells were prepared from femur and tibial bone marrow of C57BL/6 mice and the concentration was adjusted at 2 x 106/mL. BMM cells were cultured in RPMI 1640 lacking phenol red supplemented with 40 ng/mL of mouse M-CSF, 2 mM glutamine, 100 U penicillin/ 0.1mg streptomycin/mL, 10 mM HEPES buffer, 10 of charcoal-dextran-treated FBS instead of regular FCS. Total BM cells were seeded at 1 x 106 cells per mL in 2 mL of media in a 24-well plate and treated with or without NE. Various doses of NE (final concentrations, 1 x 10-8 M or 1 x 10-6 M) were added at day 0. On day 3 and day 6, half of the culture media were removed and replaced with fresh hormonedeficient media containing 40 ng/mL of M-CSF. Macrophages were harvested on day 7. To activate macrophage, 50 ng/mL of LPS was added to the culture media at day 6 and cultured for another 24 hours. For the time point study, in addition to day 0, NE (final concentration, 1 x 10-6 M) was added on day 3 and day 6. To evaluate the effects of NE on macrophage number, cells were counted immediately upon the completion of the cell culturing.Cell proliferation assayBMM proliferation regulated by NE was examined using the CyQUANT Cell Proliferation Assay Kit according to manufacturers instructions. Briefly, bone marrow cells were isolated as mentioned above and 2 x 105 BM cells were seeded into each well of a 96-well plate and treated with various doses of NE (final concentrations, 1 x 10-6 M and 1 x 10-8 M) or without NE. On day 7, media were 23977191 carefully removed, washed with PBS and plates were placed in a -80 freezer. The standard curve was made using BMMs harvested from culture flask in accordance with the protocol. Based on the standard curve, cell numbers in each sample can be calculated.Materials and MethodsReagents and AbsFITC-Dextran (MW: 40K) and LPS from Es.
