Intercellular adhesion molecule-4 and CD36 are implicated in the abnormal adhesiveness of sickle cell SAD mouse erythrocytes to endothelium

Abstract

Background: Abnormal adhesiveness of red blood cells to endothelium has been implicated in vaso-occlusive crisis of sickle cell disease. The present study examined whether the SAD mouse model exhibits the same abnormalities of red blood cell adhesion as those found in human sickle cell disease.

Design and methods: The repertoire of adhesive molecules on murine erythrocytes and bEnd.3 microvascular endothelial cells was determined by flow cytometry using monoclonal antibodies or by western blotting. Adhesion was investigated in dynamic conditions and measured at different shear stresses.

Results: CD36, CD47 and intercellular adhesion molecular-4, but not Lutheran blood group antigen/basal cell adhesion molecule, are present on mouse mature erythrocytes. alpha(4)beta(1) are not expressed on SAD and wild type reticulocytes. Endothelial bEnd.3 cells express alpha(V)beta(3), alpha(4)beta(1), CD47, vascular cell adhesion molecule-1, and Lutheran blood group antigen/basal cell adhesion molecule, but not CD36. Adhesion of SAD red cells is: (i) 2- to 3-fold higher than that of wild type red cells; (ii) further increased on platelet activating factor-activated endothelium; (iii) not stimulated by epinephrine; (iv) inhibited after treating the endothelium with a peptide reproducing one of the binding sequences of mouse intercellular adhesion molecular-4, or with mon-oclonal antibody against murine alpha(v) integrin; and (v) inhibited after pretreatment of red blood cells with anti-mouse CD36 monoclonal antibodies. The combination of treatments with intercellular adhesion molecular-4 peptide and anti-CD36 monoclonal antibodies eliminates excess adhesion of SAD red cells. The phosphorylation state of intercellular adhesion molecular-4 and CD36 is probably not involved in the over-adhesiveness of SAD erythrocytes.

Conclusions: Intercellular adhesion molecular-4/alpha(v)beta(3) and CD36/thrombospondin interactions might contribute to the abnormally high adhesiveness of SAD red cells. The SAD mouse is a valuable animal model for investigating adhesion processes of sickle cell disease.

Figure 1.

Adhesion molecules on erythrocytes of wild type and SAD mice. (A) Flow cytometry analysis of CD36, CD47, and CD147 on erythrocytes from mice bled before and after undergoing 10% O₂ hypoxia for 48 h. Results are expressed as mean fluorescence intensity (MFI). Columns represent the mean results from three means of three mice (white columns: wild-type mice; black columns: SAD mice). Bars represent SEM. **P< 0.01; ***P<0.001 by ANOVA. (B) Western blot analysis of ICAM-4 in RBC membranes. Upper panel: Mouse ICAM-4 is revealed as a 37 KD glycoprotein and a 27 KD deglycosylated protein, after digestion by N-glycosidase F (PNGase F). These bands are specific inasmuch as they were abated when the antibody was pre-incubated overnight at 4°C with a saturating concentration of the immunizing peptide or when the rabbit preimmune serum was used. Lower panel: Semi-quantitative western blot analysis of ICAM-4 expression in RBC membrane of three different SAD and wild-type C57Bl/6 mice. Equal protein loading was demonstrated by Ponceau red staining. (C) Immunoprecipitation of phosphorylated proteins from RBC membrane, followed by immunoblotting.

Figure 2.

The adhesion of SAD RBC to an endothelial monolayer is greater than that of wild-type (WT) RBC, and not altered by epinephrine (50 nM) in spite of stimulation of cAMP synthesis. (A) Adherence to endothelium in basal condition. (B) Adhesion of epinephrine-treated SAD RBC. (C) Adhesion of epinephrine-treated WT RBC. (D) Effect of epinephrine on cyclic AMP content of RBC from five SAD (black symbols and solid lines) and five WT mice (white symbols and dashed lines). Columns denote means and bars denote SEM. **P<0.01 between SAD and WT by ANOVA.

Figure 3.

Inhibition of SAD RBC adhesion on bEnd.3 endothelial cells by ICAM-4 peptide and by anti-α_V integrin monoclonal antibody. Endothelial cell monolayers on microslides were treated by 250 μM T-8-I (ICAM-4 peptide) or A-8-C (control peptide), or 10 μg/mL anti-α_V integrin antibody or control rat IgG, for 30 min before injection of RBC. (A) Adherence of SAD RBC on T-8-I peptide-treated endothelial cells. (B) Adherence of wild-type (WT) RBC on T-8-I treated endothelial cells. (C) Adherence of SAD RBC on endothelial cells treated with α_V integrin antibodies. (D) Adherence of WT RBC on endothelial cells treated with α_V integrin antibodies. ANOVA indicates that the differences observed for SAD RBC are highly significant (P< 0.001).

Figure 4.

Additive contributions of ICAM-4 and CD36 to increased adhesion of SAD RBC to endothelium. A and B: RBC were incubated in the presence 10 μg/mL monoclonal antibody anti-mouse CD36 or control mouse IgG, for 30 min. Anti-CD36 antibody significantly reduced the adhesion of SAD RBC (P<0.05) (A), but not that of wild-type (WT) RBC (B). C and D: RBC were treated with anti-CD36 mono-clonal antibody or control IgG, and bEnd.3 cell monolayers with 250 μM T-8-I or A-8-C peptide, for 30 min. The double treatment resulted in a highly significant fall of SAD RBC adhesion (C) and a moderate fall of WT RBC adhesion (D) (P<0.001 by ANOVA). Columns represent mean results from four different mice, and bars represent SEM.

Figure 5.

The adhesion of SAD RBC on PAF-activated endothelium is increased. Confluent endothelial monolayers on microslides were treated with PAF (0.2 ng/mL) for 10 min before injection of RBC. (A) At all four wall shear stresses, SAD RBC adhered in higher numbers to PAF-treated than to untreated endothelial cells (P<0.05 by ANOVA). (B) The adherence of wild-type (WT) RBC on PAF-treated endothelium was equivalent to that on non-treated endothelium. Columns represent the mean results from six different mice and bars represent SEM.