PMID- 19229187
OWN - NLM
STAT- MEDLINE
DCOM- 20090414
LR  - 20211020
IS  - 1940-087X (Electronic)
IS  - 1940-087X (Linking)
IP  - 24
DP  - 2009 Feb 11
TI  - Analysis of physiologic E-selectin-mediated leukocyte rolling on microvascular 
      endothelium.
LID - 1009 [pii]
LID - 10.3791/1009 [doi]
AB  - E-selectin is a type-1 membrane protein on microvascular endothelial cells that 
      helps initiate recruitment of circulating leukocytes to cutaneous, bone and 
      inflamed tissues. E-selectin expression is constitutive on dermal and bone 
      microvessels and is inducible by pro-inflammatory cytokines, such as IL-1alpha/ 
      and TNF-alpha, on microvessels in inflamed tissues. This lectin receptor mediates 
      weak binding interactions with carbohydrate counter-receptor ligands on 
      circulating leukocytes, which results in a characteristic rolling behavior. 
      Because these interactions precede more stable adhesive events and diapedesis 
      activity, characterization of leukocyte rolling activity and identification of 
      leukocyte E-selectin ligands have been major goals in studies of leukocyte 
      trafficking and inflammation and in the development of anti-inflammatory 
      therapeutics (1-5). The intent of this report is to provide a visual, 
      comprehensive description of the most widely-used technology for studying 
      E-selectin E-selectin ligand interactions under physiologic blood flow 
      conditions. Our laboratory in conjunction with the Harvard Skin Disease Research 
      Center uses a state-of-the-art parallel-plate flow chamber apparatus accompanied 
      by digital visualization and new recording software, NIS-Elements. This 
      technology allows us to analyze adhesion events in real time for onscreen 
      visualization as well as record rolling activity in a video format. Cell adhesion 
      parameters, such as rolling frequency, shear resistance and binding/tethering 
      efficiency, are calculated with NIS-Elements software, exported to an Excel 
      spreadsheet and subjected to statistical analysis. In the demonstration presented 
      here, we employed the parallel-plate flow chamber to investigate 
      E-selectin-dependent leukocyte rolling activity on live human bone marrow 
      endothelial cells (hBMEC). Human hematopoietic progenitor KG1a cells, which 
      express a high level of E-selectin ligand, were used as our leukocyte model, 
      while an immortalized hBMEC cell line, HBMEC-60 cells, was used as our 
      endothelial cell model (6). To induce and simulate native E-selectin expression 
      in the flow chamber, HBMEC-60 cells were first activated with IL-1 . Our video 
      presentation showed that parallel-plate flow analysis is a suitable method for 
      studying physiologic E-selectin-mediated leukocyte rolling activities and that 
      functional characterization of leukocyte E-selectin ligand(s) in the flow chamber 
      can be ascertained by implementing protease or glycosidase digestions.
FAU - Wiese, Georg
AU  - Wiese G
AD  - Department of Dermatology, Brigham and Women's Hospital.
FAU - Barthel, Steven R
AU  - Barthel SR
FAU - Dimitroff, Charles J
AU  - Dimitroff CJ
LA  - eng
GR  - R01CA118124/CA/NCI NIH HHS/United States
GR  - R01 CA118124-03/CA/NCI NIH HHS/United States
GR  - P30 AR042689/AR/NIAMS NIH HHS/United States
GR  - R01 AT004628-01A1/AT/NCCIH NIH HHS/United States
GR  - R01 AT004628/AT/NCCIH NIH HHS/United States
GR  - R01 CA118124/CA/NCI NIH HHS/United States
PT  - Journal Article
PT  - Research Support, N.I.H., Extramural
PT  - Research Support, Non-U.S. Gov't
PT  - Video-Audio Media
DEP - 20090211
PL  - United States
TA  - J Vis Exp
JT  - Journal of visualized experiments : JoVE
JID - 101313252
RN  - 0 (E-Selectin)
SB  - IM
MH  - E-Selectin/*physiology
MH  - Endothelial Cells/*cytology
MH  - Humans
MH  - Leukocyte Rolling/*physiology
MH  - Leukocytes/*cytology
PMC - PMC2730781
MID - NIHMS105667
EDAT- 2009/02/21 09:00
MHDA- 2009/04/15 09:00
PMCR- 2009/02/11
CRDT- 2009/02/21 09:00
PHST- 2009/02/21 09:00 [entrez]
PHST- 2009/02/21 09:00 [pubmed]
PHST- 2009/04/15 09:00 [medline]
PHST- 2009/02/11 00:00 [pmc-release]
AID - 1009 [pii]
AID - 10.3791/1009 [doi]
PST - epublish
SO  - J Vis Exp. 2009 Feb 11;(24):1009. doi: 10.3791/1009.