SS RBCs, but not normal RBCs, induced increased PBMC adhesion to HUVECs. (A,B) Sham-treated (A) or epinephrine (epi)–treated (B) SS RBCs (green) were coincubated with PBMCs (red). PBMC-RBC mixtures were then tested for adhesion to HUVECs. Photomicrographs using ×20 magnification show that sham-treated SS RBCs (A), which adhered to some degree to nontreated HUVECs, induced adhesion of PBMCs to HUVECs. Epinephrine-treated SS RBCs (B), which adhered strongly to nonactivated HUVECs, also induced PBMC adhesion to HUVECs. Photomicrograph of adhesion of PBMCs (not coincubated with SS RBCs) is not shown because such PBMCs did not visibly adhere to HUVECs. (C) PBMCs were tested for adhesion to HUVECs in the presence of sham- or epi-treated SS RBCs, or after lysis of sham- or epi-treated SS RBCs. Results are presented as percentage of adherent PBMCs at a shear stress of 1 dyne/cm². Error bars show SEM of 3 different experiments. *P < .001 compared with unstimulated PBMCs. (D) PBMCs separated from blood obtained from 2 different donors (donor 1, lanes 1 and 2; donor 2, lanes 3 and 4) were analyzed alone (lanes 1 and 3) or after coincubation with ABO-matched SS RBCs (lanes 2 and 4). For PBMC-RBC mixtures, after 30 minutes of incubation, cells were treated with RBC lysis buffer, then washed free of lysed RBCs. Proteins (50 μg protein per lane) obtained from both types of PBMC preparations, as well as RBCs only (lane 5), were then analyzed for the presence of RBC proteins by Western blot using antiglycophorin C and anti-CD44 (a positive control for both RBCs and leukocytes) antibodies, and P3 myeloma protein as a negative control (data not shown). Western blot analysis showed that, after RBC lysis, leukocyte preparations were free of detectable RBC proteins. (E) PBMCs not coincubated with SS RBCs did not significantly adhere to HUVECs previously coincubated with SS RBCs for 15 minutes. Similarly, the supernatant potentially containing free heme and reactive oxygen species obtained from lysed SS RBCs did not induce PBMC adhesion to ECs. Error bars show SEM of 3 different experiments. *P < .001 compared with unstimulated PBMCs. (F) Separation of SS reticulocytes (retic) and mature SS RBCs was accomplished using anti–transferrin receptor mAb 5E9 and goat anti–mouse IgG-coated magnetic microbeads. PBMCs were coincubated with sham-treated mature SS RBCs, epi-treated mature SS RBCs, sham-treated SS retic, epi-treated SS retic, or epi-treated unseparated SS RBCs. Adhesion of PBMCs to HUVECs was then tested. Results are presented as percentage of adherent PBMCs at a shear stress of 1 dyne/cm². Error bars show SEM of 3 different experiments. *P < .05 compared with unstimulated PBMCs; **P < .001 compared with PBMCs coincubated with sham-treated mature SS RBCs; ***P < .001 compared with PBMCs coincubated with sham-treated SS retic. (G) Adhesion of PBMCs to HUVECs after coincubation with sham- or epi-treated normal RBCs versus sham- or epi-treated SS RBCs, respectively. One representative experiment is presented (n = 3).