Sickle monocytes, but not lymphocytes, activate HUVEC E-selectin expression.

Sickle monocytes and lymphocytes were separated by rate zonal density gradient centrifugation using OptiPrep medium as described in “Materials and methods.” HUVEC were incubated with TNF-α (10 ng/mL) or purified sickle monocytes or lymphocytes (2 leukocytes/endothelial cell) for increasing lengths of time, from 5 minutes to 5 hours. After the indicated incubation time, the TNF-α, monocytes, or lymphocytes were removed from the HUVEC by washing and were replaced with fresh media. All HUVEC were incubated for a total of 5 hours at 37°C, including the incubation time with TNF-α or leukocytes. After the 5-hour incubation, the endothelial cells were washed and E-selectin protein expression was measured by enzyme immunoassay. Results are expressed as a percentage of control (media only) HUVEC (100%) and are from a single representative experiment of 2.