P-selectin glycoprotein ligand-1 inhibition blocks increased leukocyte-endothelial interactions associated with sickle cell disease in mice

To the editor:

Sickle cell disease (SCD) is a hemoglobinopathy caused by a single amino acid change in the β globin subunit that predisposes hemoglobin to abnormal polymerization leading to stiff, sickled erythrocytes, and microvascular occlusions.¹  Although the vascular consequences of SCD are triggered by changes in the erythrocyte,²  a chronic inflammatory state often ensues with many other cell types contributing to SCD pathology. For example, sickling of erythrocytes in the microvasculature is accompanied by increased platelet and leukocyte-endothelial (L-E) interactions that are strongly associated with vascular complications³  including stroke, priapism, leg ulcers, and nephropathy.⁴  Therapeutic targeting of molecules responsible for mediating these adhesive interactions has been shown to be potentially beneficial in preclinical models of SCD.⁵  Inhibition of P-selectin in particular has been associated with reduced L-E interactions in mouse models of SCD.⁶  The leukocyte ligand for P-selectin is P-selectin glycoprotein ligand-1 (Psgl-1), which also interacts with E- and L-selectin.

Psgl-1 inhibition may be particularly effective in reducing adhesive interactions in SCD since Psgl-1–mediated signaling also affects activation of leukocytes,⁷  which in turn affects adhesive properties of the endothelium.⁸  To determine the effectiveness of Psgl-1 inhibition on reduction of L-E interactions in the microvasculature, we studied a humanized mouse model of SCD⁹  treated with an anti–mouse Psgl-1 antibody. First, C57BL/6J mice underwent a bone marrow transplantation (BMT) procedure as previously described.⁸  Recipient mice received bone marrow from wild-type donors (Hbb^+/+) or donors homozygous for the sickle cell mutation (Hbb^hβs/hβs). Eight weeks after BMT, mice receiving Hbb^hβs/hβs marrow (n = 10) were anemic compared with mice receiving Hbb^+/+ marrow (n = 10; hematocrit - 28.9 ± 1.0 vs 36.7 ± 0.8%, P < .0001), (hemoglobin - 10.0 ± 0.5 vs 12.3 ± 0.4 g/dL, P < .001) and (RBC counts 7.5 ± 0.5 vs 9.1 ± 0.2 × 10⁶/μL, P < .01). Sickle-shaped erythrocytes were frequent in the peripheral blood smears of Hbb^hβs/hβs mice and Hbb^hβs/hβs mice exhibited a marked reticulocytosis compared with Hbb^+/+ mice (reticulocyte - 30.2 ± 4.4 vs 3.4 ± 0.6% of RBCs, P < .00001). At the time of sacrifice (20 weeks after BMT), the average spleen size of Hbb^hβs/hβs mice was 370.7 ± 55.4 mg compared with 74.9 ± 2.4 mg in Hbb^+/+ mice, P < .000001. At 16 weeks post BMT, mice were treated with weekly IV injection of anti–Psgl-1 antibody or control IgG antibody for 4 weeks. Intravital microscopy⁸  was performed 20 weeks following BMT using intravenous rhodamine to label leukocytes. Leukocyte rolling and firm attachment were increased in Hbb^hβs/hβs mice compared with Hbb^+/+ mice. However, anti–Psgl-1 treatment completely reversed the increased leukocyte rolling and firm attachment within the Hbb^hβs/hβs mice. (Figure 1A-D). Reduced L-E interactions were associated with reductions in levels of circulating soluble E-selectin (sE-sel), soluble P-selectin (sP-sel), and soluble vascular cell adhesion molecule 1(sVCAM-1; Figure 1G-I). Four weeks of treatment was sufficient to reduce iron deposition and necrotic areas of liver (Figure 1L,O). This was associated with reduced CD68 and TNF-α expression in the liver by RT-PCR (Figure 1E-F). In conclusion, inhibition of Psgl-1 may be an effective treatment for reducing vascular complications of SCD.