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We reported that complement cascade (CC) becomes activated in bone marrow (BM) during granulocyte colony stimulating factor (G-CSF) mobilization of hematopoietic stem/progenitor cells (HSPCs) and demonstrated that while the third CC component (C3)-deficient mice are easy mobilizers, the fifth CC component (C5)-deficient mice mobilize very poorly. To explain this, we postulated that activation/cleavage of CC releases C3a and C5a anaphylatoxins that differently regulate mobilization. Accordingly, C3a, by enhancing responsiveness of HSPCs to decreasing concentrations of stromal-derived growth factor-1 (SDF-1) in BM, prevents mobilization and promotes their BM-retention. As such, we focused on the mobilization-enhancing role of C5a herein. We found that C5a receptor (C5aR) is not expressed on the surface of HSPCs, and C5a-mediated pro-mobilization effects are mediated by stimulation of granulocytes. Overall, our data support a following model. First C5aR+ granulocytes are chemoattracted by plasma C5 cleavage fragments, being the first wave of cells leaving BM. This facilitates subsequent egress of HSPCs. In the next step, after leaving the BM, granulocytes undergo degranulation in response to plasma C5a and secrete some cationic peptides (cathelicidin, β-defensin) that as demonstrated here for a first time highly enhance responsiveness of HSPCs to plasma SDF-1 gradient. In conclusion, our data reveal the underappreciated central role of innate immunity in mobilization where C5 cleavage fragments via granulocytes orchestrate this process.

Study design: Experimental laboratory investigation of the role and pathways of reactive oxygen species (ROS)-mediated motor neuron cell death in a mouse model of compression spinal cord injury. Objectives: To analyze ROS-mediated oxidative stress propagation and signal transduction leading to motor neuron apoptosis induced by compression spinal cord injury. Setting: University of Louisville Health Science Center. Methods: Adult C57BL/6J mice and transgenic mice overexpressing SOD1 were severely lesioned at the lumbar region by compression spinal cord injury approach. Fluorescent oxidation, oxidative response gene expression and oxidative stress damage markers were used to assay spinal cord injury-mediated ROS generation and oxidative stress propagation. Biochemical and immunohistochemical analyses were applied to define the ROS-mediated motor neuron apoptosis resulted from compression spinal cord injury. Results: ROS production was shown to be elevated in the lesioned spinal cord as detected by fluorescent oxidation assays. The early oxidative stress response markers, NF-kB transcriptional activation and c-Fos gene expression, were significantly increased after spinal cord injury. Lipid peroxidation and nucleic acid oxidation were also elevated in the lesioned spinal cord and motor neurons. Cytochrome c release, caspase-3 activation and apoptotic cell death were increased in the spinal cord motor neuron cells after spinal cord injury. On the other hand, transgenic mice overexpressing SOD1 showed lower levels of steady-state ROS production and reduction of motor neuron apoptosis compared to that of control mice after spinal cord injury. Conclusion: These data together provide direct evidence to demonstrate that the increased production of ROS is an early and likely causal event that contributes to the spinal cord motor neuron death following spinal cord injury. Thus, antioxidants/antioxidant enzyme intervention combined with other therapy may provide an effective approach to alleviate spinal cord injuryinduced motor neuron damage and motor dysfunction.