Abstract
Microvascular vaso-occlusion in sickle cell disease is thought to involve adhesive interactions among erythrocytes (RBCs), leukocytes and vascular endothelial cells. Recent studies have demonstrated the presence of a significant inflammatory response in sickle cell disease, including changes in the cell surface adhesion molecules that mediate cell-cell interactions in the microvasculature. In this study, we used a parallel-plate flow chamber assay to determine the subpopulations of leukocytes that are involved in sickle leukocyte-RBC interactions. We also studied the effect of treatment with hydroxyurea (HU) on these adhesive interactions. Populations of monocytes, neutrophils (PMNs) and T cells were isolated by negative selection from the peripheral blood of untreated patients with sickle cell disease (SS), sickle patients receiving HU (SS-HU), and healthy control subjects (AA). Adhesive interactions involving these leukocyte subpopulations, human umbilical vein endothelial cells (HUVECs) pretreated with tumor necrosis factor-α (TNF-α ), and autologous RBCs were measured under a shear stress of 1 dyne/cm2. Compared to the corresponding cell populations from AA individuals, PMNs, monocytes, and T cells from SS individuals were significantly more adherent to TNF-α-treated HUVECs (774±59 vs. 502±27 cells/mm2, p=0.001; 533±66 vs. 348±36 cells/mm2, p=0.024; and 470±75 vs. 227±26 cells/mm2, p=0.009, respectively). HU therapy significantly decreased the adhesion of SS PMNs to HUVECs (774±59 cells/mm2 for SS vs. 604±36 for SS-HU, p=0.025). Compared to adherent AA leukocytes, adherent SS leukocytes exhibited greater participation in adhesive interactions with autologous RBCs (41±3% for SS vs. 27±3% for AA, p=0.002), and HU treatment decreased the fraction of leukocytes that captured autologous RBCs to the control level (29±3% for SS-HU, p=0.006 vs. SS). Compared to adherent PMNs from SS individuals, adherent PMNs from SS-HU individuals showed significantly reduced participation in the capture of RBCs (53±6% for SS vs. 35±5% for SS-HU, p=0.021). Although adherent T cells from SS individuals participated significantly more in RBC capture than adherent T cells from AA individuals (28±5% for SS vs. 10±2% for AA, p=0.007), HU therapy did not have a significant effect on this parameter (21±5% for SS-HU, p=0.373). Compared to AA leukocytes, SS leukocytes captured more RBCs per participating adherent leukocyte (2.8±0.2 vs. 1.9±0.1 RBCs/cell, p=0.001). HU therapy reduced the number of RBCs captured per PMN but not the number captured per T cell. Compared to AA T cells, SS T cells captured adherent RBCs for a significantly longer period of time (51±9 vs. 26±6 seconds, p=0.035). Our data suggest that sickle neutrophils, monocytes and T cells may all be involved in adhesive interactions with sickle RBCs. PMN-RBC and monocyte-RBC interactions appear to be more numerous than T cell-RBC interactions, although T cell-RBC interactions may be stronger. HU therapy appears to target PMN-RBC and monocyte-RBC interactions preferentially. Future studies will focus on the role of particular adhesion molecules in mediating these interactions and on the potential for therapeutic interventions targeting cell-cell adhesion.
Tuning intercellular adhesion with membrane-anchored oligonucleotides
