Dynamic Imaging of Marrow-Resident Granulocytes Interacting with Human Mesenchymal Stem Cells upon Systemic Lipopolysaccharide Challenge
Figure 3
Time-lapse TPM images of LysM+ granulocytes interacting with hMSC after systemic LPS administration. CTO-labeled hMSCs (red) were injected i.v. into a recipient mouse containing LysM+ (green) granulocytes 20 hours prior to performing intravital TPM of the bone marrow through intact calvarium of the live recipient mouse. The recipient mouse received a 100 ng of LPS injection i.v. at relative time = 0 min ( = 0′). (a) Sequential TPM images from Supplemental Movie 1 (See Supplementary Materials available online at http://dx.doi.org/10.1155/2013/656839) displayed at 10-minute intervals for a total of 110 minutes ( = 110′) starting from the time of LPS injection ( = 0′), showing the accumulation of LysM+ GFP+ granulocytes surrounding 2 out of 3 hMSCs (red) that are clearly visible within the imaging field. Scale bar = 20 μm. (b) Total fluorescence of GFP signal within a 20 μm radius of each of the three hMSCs identified in (a) at = 110′ over time. (c) Time-averaged percent of LysM+ GFP+ neutrophils within 20 μm radius of the hMSC that formed a cell-cell contact with the said hMSCs after LPS challenge. The percent was normalized to the contact frequency at = 0′. (d) Track analysis of cell number 3 in (a) = 110′. Shown are migration tracks of surrounding LysM+ GFP+ granulocytes during 0 to 30 minutes (left panel) and 60 to 90 minutes (right panel) after LPS administration. Individual migration heat map tracks are color-coded based on the calculated instantaneous speed. Yellow sphere = 20 μm radius around the center of hMSC number 3. Scale bar = 50 μm. (e) Cell flux indices for 3 different hMSCs identified in (a) are shown, where all three hMSCs showed net increased neutrophil flux towards them after LPS administration, although hMSC number 3 exhibited heightened and sustained positive cellular flux. hMSC number 2 had an initial increase in positive flux, which reached homeostasis two hours after LPS injection. hMSC number 1 initially had a negative cellular flux due to local neutrophils being attracted to hMSCs number 2 and number 3. However, neutrophils from regions beyond the imaging field began to migrate towards the hMSC 30 to 60 minutes following LPS injection before the cellular traffic reached a steady-state level (cell flux index of ~ 0).
