Bone marrow norepinephrine mediates development of functionally different macrophages after thermal injury and sepsis

Abstract

Objective: We sought to determine the influence of thermal (burn) injury with sepsis and norepinephrine on the clonogenic potential and functional cytokine response to lipopolysaccharide (LPS) stimulation in nonmyeloid committed (CD117) and myeloid committed (ER-MP12) bone marrow progenitor cells.

Summary and background data: We have previously demonstrated that norepinephrine stimulated myelopoiesis after burn injury and sepsis, but the site of this stimulation in monocyte development is unknown. In the present study the influence of norepinephrine on the developmental hierarchy of bone marrow cells after thermal injury and sepsis was determined by assessing the clonogenic potential and LPS-stimulated cytokine responses of mature macrophages derived from CD117 and ER-MP12 bone marrow progenitor cells.

Methods: Tissue and bone marrow norepinephrine content was ablated by chemical sympathectomy with 6-hydroxydopamine treatment. CD117 and ER-MP12 bone marrow cells were isolated using antibody-linked magnetic microbeads. Clonogenic potential in response to colony-stimulating factors was determined. Both progenitor cell types were differentiated to mature macrophages in vitro and tumor necrosis factor (TNF)-alpha and interleukin (IL)-6 cytokine responses to LPS provocation were determined.

Results: The macrophage- and granulocyte-macrophage colony-stimulating factor responsive clonogenic potential was increased with burn sepsis, suggesting an expansion of both progenitor populations. Such increases were greatly reduced with prior depletion of norepinephrine. TNF-alpha and IL-6 cytokine responses to LPS were markedly influenced by the specific progenitor cells involved as well as the injury conditions and the status of norepinephrine prior to injury. In burn sepsis the depletion of norepinephrine resulted in a dramatic decrease in both IL-6 and TNF-alpha production by both progenitor-derived macrophages.

Conclusions: Depletion of norepinephrine attenuated burn and burn sepsis-induced bone marrow progenitor clonal growth in response to macrophage- and granulocyte-macrophage colony-stimulating factor. Functional phenotypes of bone marrow progenitor-derived macrophages are greatly influenced by norepinephrine and the milieu created by thermal injury and sepsis.

FIGURE 1. Dual-color flow cytometric analysis for the expression of CD34 and CD117 antigens on total nucleated bone marrow cells after sham, burn, and burn sepsis 72 hours after injury in norepinephrine-intact (NE intact) and norepinephrine-depleted (NE depleted) animals. X- and Y-axes represent fluorescent intensity in log scale. Each panel is representative of 4 animals in each group.

FIGURE 2. Soft agar clonogenic assay for CD117⁺ bone marrow progenitors 72 hours after sham, burn, and burn sepsis in norepinephrine intact (A) and norepinephrine depleted (B) animals. CD117⁺ cells (2.5 × 10⁴ cells/mL) were incubated in RPMI media containing 0.3% agar in the presence of rmIL-3 and rmFlt3 with either rmGM-CSF or rmM-CSF (all at 10 ng/mL). Colonies (>50 cells) were counted with a light microscope after 7 days of incubation. All assays were done in triplicate and each bar represents results of 3 experiments (n = 3) involving 6–15 animals each. *P < 0.05 versus sham; #P < 0.05 versus burn.

FIGURE 3. Soft agar clonogenic assay for ER-MP12⁺ bone marrow progenitors 72 hours after sham, burn, and burn sepsis in norepinephrine intact (A) and norepinephrine depleted (B) animals. ER-MP12⁺ cells (2.5 × 10⁴ cells/mL) were incubated in RPMI media containing 0.3% agar in the presence of rmIL-3 and rmFlt3 with either rmGM-CSF or rmM-CSF (all at 10 ng/mL). Colonies (>50 cells) were counted with a light microscope after 7 days of incubation. All assays were done in triplicate and each bar represents results of 3 experiments (n = 3) involving 6–15 animals each. *P < 0.05 versus sham; # P < 0.05 versus burn.

FIGURE 4. LPS-stimulated IL-6 production (A) and TNF-α production (B) in CD117⁺ and ER-MP12⁺ bone marrow progenitor-derived macrophage taken from animals with intact norepinephrine. Progenitor cells were isolated from sham, burn and burn sepsis mice and differentiated into macrophages with a cocktail of growth factors (rmFlt-3 Ligand, rmIL-3, rmM- and rmGM-CSF all 10 ng/ml) for 7 days at 37°C. Macrophages were stimulated with LPS (100ng/ml) for 18 hours and cytokines were measured in the supernatants and normalized to cellular protein concentration. All assays were done in triplicate and each bar represents results of 3 experiments (N=3) involving 6–15 animals each. *P < 0.05 versus sham; #P < 0.05 versus burn.

FIGURE 5. LPS-stimulated IL-6 production (A) and TNF-α production (B) in CD117⁺ bone marrow progenitor-derived macrophage taken from animals with and without norepinephrine depletion. Progenitor cells were isolated from sham, burn and burn sepsis mice with intact or depleted norepinephrine. CD117⁺ progenitors were differentiated into macrophages with a cocktail of growth factors (rmFlt-3 Ligand, rmIL-3, rmM- and rmGM-CSF all 10 ng/ml) for 7 days at 37°C. Macrophages were stimulated with LPS (100ng/ml) for 18 hours and cytokines were measured in the supernatants and normalized to cellular protein concentration. All assays were done in triplicate and each bar represents results of 3 experiments (n = 3) involving 6–15 animals each. *P < 0.05 versus NE intact.

FIGURE 6. LPS-stimulated IL-6 production (A) and TNF-α production (B) in ER-MP12⁺ bone marrow progenitor-derived macrophage taken from animals with and without norepinephrine depletion. Progenitor cells were isolated from sham, burn and burn sepsis mice with intact or depleted norepinephrine. ER-MP12⁺ progenitors were differentiated into macrophages with a cocktail of growth factors (rmFlt-3 Ligand, rmIL-3, rmM- and rmGM-CSF all 10 ng/ml) for 7 days at 37°C. Macrophages were stimulated with LPS (100 ng/mL) for 18 hours and cytokines were measured in the supernatants and normalized to cellular protein concentration. All assays were done in triplicate and each bar represents results of 3 experiments (n = 3) involving 6–15 animals each. *P < 0.05 versus NE intact.