Clinical observations have suggested a role for leukocytes in the pathophysiology of sickle cell disease (SCD). We have previously shown that epinephrine (epi), a stress hormone, activates LW-mediated sickle red cell (SS RBC) adhesion to non-activated endothelial cells (ECs). LW also recognizes leukocyte integrins. Moreover, SS RBCs can activate ECs. Therefore, we hypothesized that interaction of epi-activated LW on SS RBCs with leukocytes may also induce activation of leukocytes and stimulate their adhesion to ECs. To evaluate our hypothesis, SS RBCs were sham-treated or treated with 20 nM epi for 1 min, and after three washes, were incubated for 30 min with normal mononuclear leukocytes at 37°C. SS RBCs in leukocyte-RBC mixtures were lysed before measurement of leukocyte adhesion to non-activated ECs. Leukocytes not pre-incubated with SS RBCs and leukocytes treated with RBC lysis buffer alone did not adhere to ECs at a shear stress of 1 dyne/cm2. Incubation of sham-treated SS RBCs or sham- or epi-treated normal RBCs with leukocytes induced a non-significant adherence of 10±3%, 0% and 2% of the leukocyte population, respectively. Epi-treated SS RBCs, however, induced adhesion of 42±4.8% of leukocytes to ECs (p < 0.001). When sham- or epi-treated SS RBCs in RBC-leukocyte mixtures were not lysed, leukocyte adhesion to ECs did not further increase when compared to adhesion of leukocytes washed free of lysed sham-treated or epi-treated SS RBCs. The possible effect of residual epi on leukocytes was also tested. Exposure of leukocytes to the supernatant of the last wash of SS RBC treatment with epi had no effect on leukocyte adhesion to ECs, compared to adhesion of non-treated leukocytes to ECs. We next determined whether activated SS RBCs interact with leukocytes via the RBC LW glycoprotein. Pre-incubation of SS RBCs with antibody to LW (25 μg/ml) reduced adhesion of leukocytes induced by epi-treated SS RBCs by 78±7.9 % (p < 0.001), while pre-incubation of SS RBCs with antibody to the CD47 thrombospondin receptor (25 μg/ml) failed to inhibit the ability of epi-treated SS RBCs to stimulate leukocyte adhesion to ECs. We also identified the molecule on leukocytes mediating adhesion to ECs and its counterreceptor on ECs. Antibody to the CD44 adhesion receptor (50 μg/ml) on leukocytes blocked adhesion to ECs of leukocytes activated by epi-treated SS RBCs by 71±6.35% (p < 0.05), while antibody to CD47 failed to inhibit adhesion of activated leukocytes to ECs. Similarly, addition of recombinant soluble (rs) CD44 protein (50 μg/ml) to ECs also blocked adhesion of activated leukocytes to ECs by 74±5.8 % (p < 0.01). In contrast, incubation of rsLW (50 μg/ml) with ECs failed to inhibit adhesion of activated leukocytes. To define the endothelial counterreceptor for CD44, we incubated ECs with antibodies to adhesion molecules αVβ3 integrin, fibronectin, thrombospondin, CD44, E-selectin or P-selectin before adhesion assays. Only antibody to endothelial E-selectin completely blocked adhesion of activated leukocytes to ECs. Flow cytometric analysis showed that 36% of cultured ECs highly expressed E-selectin. These data suggest that leukocyte CD44 can be activated by cell-cell interactions mediated by RBC LW to stimulate adhesion of leukocytes to endothelial E-selectin. These studies thus provide a previously unsuspected mechanism whereby activated SS RBCs may play a role as potent stimulatory cells capable of activating leukocyte adhesion to endothelium in SCD.

Disclosure: No relevant conflicts of interest to declare.

Author notes


Corresponding author