Aggregation of mononuclear and red blood cells through an {alpha}4{beta}1-Lu/basal cell adhesion molecule interaction in sickle cell disease

Abstract

Background: Abnormal interactions between red blood cells, leukocytes and endothelial cells play a critical role in the occurrence of the painful vaso-occlusive crises associated with sickle cell disease. We investigated the interaction between circulating leukocytes and red blood cells which could lead to aggregate formation, enhancing the incidence of vaso-occlusive crises.

Design and methods: Blood samples from patients with sickle cell disease (n=25) and healthy subjects (n=5) were analyzed by imaging and classical flow cytometry after density gradient separation. The identity of the cells in the peripheral blood mononuclear cell layer was determined using antibodies directed specifically against white (anti-CD45) or red (anti-glycophorin A) blood cells.

Results: Aggregates between red blood cells and peripheral blood mononuclear cells were visualized in whole blood from patients with sickle cell disease. The aggregation rate was 10-fold higher in these patients than in control subjects. Both mature red blood cells and reticulocytes were involved in these aggregates through their interaction with mononuclear cells, mainly with monocytes. The size of the aggregates was variable, with one mononuclear cell binding to one, two or several red blood cells. Erythroid Lu/basal cell adhesion molecule and α(4)β(1) integrin were involved in aggregate formation. The aggregation rate was lower in patients treated with hydroxycarbamide than in untreated patients.

Conclusions: Our study gives visual evidence of the existence of circulating red blood cell-peripheral blood mononuclear cell aggregates in patients with sickle cell disease and shows that these aggregates are decreased during hydroxycarbamide treatment. Our results strongly suggest that erythroid Lu/basal cell adhesion molecule proteins are implicated in these aggregates through their interaction with α(4)β(1) integrin on peripheral blood mononuclear cells.

Figures

Figure 1.

Abnormal SS RBC co-selection during PBMC density gradient separation. (A) Typical histograms representing flow cytometry analysis of the PBMC layer in one AA subject (left panels) and one SS patient (right panels). Upper and lower panels represent CD45-FITC (PBMC) and GPA-PE (RBC) staining, respectively. The horizontal lines represent areas of positive events; percentages indicate the proportions of positive events for each marker. (B) RBC percentage in the PBMC layer in AA subjects (▪) (n=5) and SS patients (▴) (n=17). Horizontal lines indicate medians. The percentage of RBC in the PBMC layer is significantly higher in SS patients than in AA subjects. *P=0.0013, Mann-Whitney test.

Figure 2.

SS patients have abnormally high rates of RBC-PBMC aggregates. (A) Typical dot plot representation of the PBMC layer flow cytometry analysis in one AA subject (left panel) and one SS patient (right panel). CD45-FITC and GPA-PE antibodies stain PBMC and RBC, respectively. CD45-FITC and GPA-PE double-stained events represent potential RBC-PBMC aggregates. (B) Imaging flow cytometry showing dot plots of one SS patient (upper left panel) and one AA subject (lower left panel), as in (A). Right panels show typical examples of brightfield and fluorescent images from the area of double-stained events. Fluorescent images show, from left to right, CD45-FITC-labeled PBMC, GPA-PE-labeled RBC or RBC-derived microparticles and CD45-FITC/GPA-PE double-stained events. The double-stained area was divided into four parts according to the RBC:PBMC ratio in aggregates: >2:1 (P1), 2:1 (P2), 1:1 (P3). The fourth part shows the presence of PBMC interacting with RBC-derived microparticles (P4). Double-stained events from the AA subject were essentially restricted to PBMC interacting with RBC-derived microparticles (P5). (C) Percentages of PBMC involved in aggregates in AA subjects (▪) (n=5) and SS patients (▴) (n = 17). Horizontal lines indicate medians. *P=0.0048, Mann-Whitney test.

Figure 3.

SS RBC and PBMC populations involved in aggregation. (A) Aggregates include both mature SS RBC and reticulocytes. Typical results showing cells from the PBMC layer labeled with FITC-conjugated anti-CD45, PE-conjugated anti-GPA and APC-conjugated anti-CD71 antibodies. The dot plot representation in the left panel shows gated events corresponding to SS RBC, PBMC and aggregates. Each of these three populations was gated and analyzed for CD71 expression, as shown in the middle panel. CD71 expression in aggregates is magnified in the right panel. The bold horizontal lines represent CD71-positive events. (B) Monocytes have a greater ability to aggregate than lymphocytes. The dot plot representation in the left panel is as for (A). Lymphocytes (L) and monocytes (M) were distinguished among PBMC and aggregates using morphological parameters (forward versus side scatter, right panels). (C) Percentage of lymphocytes (□) and monocytes (▴) in the PBMC population and in aggregates. Horizontal lines indicate medians.

Figure 4.

α4β1 integrin and Lu/BCAM are involved in RBC-PBMC aggregation. Inhibition assays using VCAM-1-Fc (A) or anti-β1 antibody (B). *P=0.03, Wilcoxon's matched-pairs test. (C) The dot plot representation in the left panel shows gated events corresponding to SS RBC, PBMC and aggregates. Each of these three populations was gated and analyzed for Lu/BCAM expression, as shown in the middle panel. Lu/BCAM expression in aggregates is magnified in the right panel. Bold horizontal lines represent Lu/BCAM-positive events. (D) Percentage of Lu/BCAM-positive events in the RBC population (▴) and in aggregates (▾) of five SS patients. Horizontal lines indicate medians.

Figure 5.

Hydroxycarbamide (HC) decreases the rate of RBC-PBMC aggregation in SS patients. Percentage of PBMC in aggregates in SS untreated patients (▪, n=17), HC-treated patients (▴, n=8) and AA subjects (▾, n=5). *P=0.013, **P=0.0186, Mann-Whitney test.