Primary role for adherent leukocytes in sickle cell vascular occlusion: a new paradigm

Abstract

Vascular occlusion is the major cause of morbidity and mortality in sickle cell disease but its mechanisms are poorly understood. We demonstrate by using intravital microscopy in mice expressing human sickle hemoglobin (SS) that SS red blood cells (RBCs) bind to adherent leukocytes in inflamed venules, producing vasoocclusion of cremasteric venules. SS mice deficient in P- and E-selectins, which display defective leukocyte recruitment to the vessel wall, are protected from vasoocclusion. These data uncover a previously unsuspected paradigm for the pathogenesis of sickle cell vasoocclusion in which adherent leukocytes play a direct role and suggest that drugs targeting SS RBC-leukocyte or leukocyte-endothelial interactions may prevent or treat the vascular complications of this debilitating disease.

Figure 1

Analysis of donor chimerism by Hb electrophoresis. Hb lysates were electrophoresed (20 μg per lane) on an acidic polyacrylamide gel containing urea and Triton X-100. The m Hbβ (lane 1) and h Hbβ^S (lane 2) comigrate, but m Hbα and h Hbα can be distinguished. Various m:h Hb mixtures (lanes 3–7) were used to define the sensitivity of detecting residual m Hb (≈1.5%). Three representative (*) wild-type mice that received SS bone marrow demonstrated >97% donor h Hb. Only mice with >97% h Hb were used in the studies.

Figure 2

Analyses of leukocyte and SS RBC adhesion events in postcapillary and collecting venules of SS, SA, and WT mice. (A) The numbers of rolling and adherent leukocytes were determined from video sequences recorded between 15 and 90 min after surgery. The numbers of rolling and adherent leukocytes were significantly higher in the venules of SS mice compared with WT controls; *, P < 0.05; #, P < 0.001. (B) SS RBC interactions with adherent leukocytes were determined in venules recorded between 30 and 90 min after the cremasteric surgery. A mean of 1.7 RBCs interacted per adherent leukocyte per minute in SS mice, whereas these interactions were virtually absent in SA or WT mice; ¥, P < 0.0001. (C) Number of RBC–WBC interactions during IVM 1 in SS mice. Each dot represents an individual venule. The number of SS RBC interactions per adherent leukocyte per minute, evaluated over 100 μm venular lengths during IVM 1, increased with time after cremasteric surgery (R = 0.46; n = 44 venules; P = 0.002). (D) Wall shear rates (γ) in venules of SS, SA, and WT mice during IVM 1 and IVM 2; ¶, P < 0.0001. Data are mean ± SE; n = 25–36 venules from four to five mice.

Figure 3

Intravital microscopy of the cremasteric microcirculation in SA- and SS-transplanted mice. (A) Still frame of a venule from an SA-transplanted mouse after TNF-α injection, during IVM 2. Numerous rolling leukocytes (round cells) and some adherent leukocytes can be observed, but RBCs flow freely. Owing to the rapid blood flow, RBCs cannot be distinguished in the microcirculation of live animals unless they are interacting. (B) Venule of a SS transplanted mouse during IVM 2. Numerous SS RBCs (arrows) bind to adherent leukocytes (arrowheads) and can resist the shear of flowing blood. (C) Large venule of an SS mouse after TNF-α stimulation. The parallel bars mark the venular walls. Both sickle- and discoid-shaped RBCs (arrow) bind to an adherent leukocyte (arrowhead). See Movie 1, which is published as supporting information on the PNAS web site (www.pnas.org), for the original video segments. (D–F) Sequential digital frames of a venular segment during the intravital microscopy protocol. (D) Fifteen minutes after surgery, only rolling and adherent leukocytes are observed and no RBC adhesion is seen. (E) At the end of IVM-1, many interactions between sickle cells (arrows) and adherent leukocytes are apparent. (F) Venular occlusion during IVM 2 (100 min after TNF-α). Individual blood cells cannot be easily distinguished in the multicellular aggregates of occluded venules (*), except for a few leukocytes (arrowheads) and sickle cells (arrows). (Scale bars, 15 μm.)

Figure 4

Sickle cell mice deficient in both P- and E-selectins are protected from vasoocclusion. (A) Hb gel electrophoresis of SS P/E −/− chimeras. Blood lysates from m and h controls and three SS P/E −/− chimeric mice (*) were electrophoresed on an acid/urea/Triton 100-X gel and the gel was stained with Coomassie blue. RBCs from all studied SS P/E −/− chimeric mice contained >97% h Hb. (B) The number of rolling leukocytes in SS and SS P/E −/− mice during IVM 1 and IVM 2. The number of rolling leukocytes was drastically reduced in SS P/E −/− animals compared with SS mice during IVM 1 (P = 0.0001). The reduction in the rolling leukocytes in SS mice during IVM 2 (a), compared with IVM 1, results in part from the lower wall shear rates due to vascular occlusions in the venules of SS mice (see Fig. 2D). (C) Numbers of adherent leukocytes in SS and SS P/E −/− animals during IVM 1 and IVM 2. The number of adherent leukocytes was significantly reduced in SS P/E −/− compared with SS animals both during IVM 1 and IVM 2 (P < 0.0001). (D) Wall shear rates in venules of SS P/E −/− mice during IVM 1 and IVM 2. In contrast to SS mice (Fig. 2D), shear rates were mildly decreased during IVM 2 (P = 0.01). (E) Still photograph of a venule from a SS P/E −/− animal 90 min after TNF-α. No rolling and one adherent leukocyte (arrowhead) can be observed. Blood flow (right to left) is preserved even in small postcapillary venules (arrows). See Movie 3.