Imaging Flow Cytometry and Microfluidic Flow Assays Demonstrate Heterocellular Aggregation Of Immature Sickle Erythrocytes To Neutrophils Via Mac-1/VLA-4 Interactions

Vaso-occlusion (VO) is a major acute complication of sickle cell disease (SCD). Sickle mouse models indicate that the recruitment of leukocytes to the endothelium may participate in the initiation/propagation of VO, consequently interacting with circulating erythrocytes and other cell types to impair blood flow. We investigated, in vitro, whether neutrophils (Neut) circulate in heterocellular aggregates with red blood cells (RBC) in sickle cell anemia (SCA) patients, and whether Neut from SCA individuals interact with RBC once tethered to vessel wall components.

Peripheral blood was obtained from healthy control individuals (CON) and SCA patients in steady state, off (SCA) and on hydroxyurea (SCAHU; 15-30mg/kg/day). After Ficoll-sedimentation of granulocytes and incubation with antibodies against RBC/Neut surface proteins (RBC: CD235a, CD71; Neut: CD66b, CD11a, CD11b), neutrophil-red blood cell aggregates (Neut-RBC agg) were evaluated using the Amnis® ImageStream^X MKII imaging flow cytometer. CD66b+ events (6000) were acquired at 40X and data analyzed by IDEAS 6.0. Neutrophil adhesion and heterotypic interactions with RBC were also monitored using a Cellix Venaflux^TM microfluidics platform, employing 800-μm-channel biochips lined with activated human umbilical vein endothelial cells (HUVECs), or E-selectin (4µg/mL).

Circulating Neut-RBC agg (CD66⁺CD235a⁺ events) were significantly increased in SCA, compared to CON individuals (Mean ±SEM % of Neut aggregated with RBC; SCA, 10.9 ±1.6%; SCAHU, 11.5 ±2.6%; CON, 5.4 ±1.7%; n≥7; p<0.05, SCA, comp. to CON) (See Figure). Neut-RBC agg were composed mainly of CD235⁺CD71⁺ reticulocytes, as opposed to mature RBC (CD235⁺CD71^-) (Mean ±SEM; SCA, 6.9 ±1.6%; SCAHU, 7.2 ±1.9%; CON, 1.8 ±1.3% Neut aggregated to reticulocytes; n≥7; p<0.01). Sizes of aggregates were variable, with each neutrophil binding to 1 or 2 or more RBCs. Fetal hemoglobin (HbF) levels in SCA patients inversely correlated with the percentage of Neut-RBC agg (r_S=-0.55; p<0.05; n=17). While the surface expressions of the neutrophil β₂ integrins, LFA-1 and Mac-1, were not significantly different between CON, SCA and SCAHU patients, Mac-1 (expressed on Neut), and VLA-4 (expressed on reticulocytes)-blocking antibodies and a peptide inhibitor of ICAM-4 (expressed on mature RBC), significantly decreased Neut-RBC agg found in SCA samples, compared to the respective isotype negative controls (INC) (Mean ±SEM; INC, 7.75 ±1.7%; anti-Mac-1, 4.9 ±1.5%, p<0.01; anti-VLA-4, 5.67 ±1.5%, p<0.05; anti-ICAM-4, 5.63 ±1.5%, p<0.05; n≥13). Using a microfluidic platform to monitor interactions of RBC with Neut adhered to vessel wall components, perfused SCA RBC were found to interact significantly more with autologous Neut previously adhered to activated HUVECs, compared to RBC from SCAHU and CON individuals (Mean ±SEM of RBC adhered/Neut: 2.5 ±0.5; 1.0 ±0.3; 1.0 ±0.3, respectively; n≥09; p=0.01). Furthermore, the duration of interactions of RBC with autologous Neut pre-adhered to recombinant E-selectin was significantly higher for SCA RBC, compared to CON RBC and RBC from patients on HU (Mean ±SEM duration of interactions: SCA, 1.57 ±0.10s; CON, 1.17 ±0.04s; SCAHU, 1.19 ±0.04s; n≥05; p<0.01 SCA, comp. to CON).

A significant proportion of neutrophils of SCA patients circulate in the form of neut-RBC agg, particularly involving reticulocytes; higher HbF levels were associated with decreased Neut-RBC agg formation. Furthermore, Neut from SCA individuals, when adhered to activated endothelial cells or endothelial ligands, interact more readily with SCA RBC; additionally, HU therapy is associated with a reduction in these heterotypic interactions. Antibodies or pharmacological approaches that reduce the formation of Neut-RBC agg by targeting Mac-1/VLA-4 interactions may hold potential for clinical benefits in SCD.

Figure

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Representative images of circulating Neut-RBC agg from CON and SCA individuals.

(A) Brightfield (BF) and respective fluorescent images: purple color – CD66b (Neut); yellow color – CD235a (RBC).

(B) BF and fluorescence staining imaging of adhesion molecules analyzed on Neut-RBC agg from an SCA individual; CD66b, CD11a, CD11b, CD235a, CD71, SSC; side scatter.

Figure

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Representative images of circulating Neut-RBC agg from CON and SCA individuals.

(A) Brightfield (BF) and respective fluorescent images: purple color – CD66b (Neut); yellow color – CD235a (RBC).

(B) BF and fluorescence staining imaging of adhesion molecules analyzed on Neut-RBC agg from an SCA individual; CD66b, CD11a, CD11b, CD235a, CD71, SSC; side scatter.

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No relevant conflicts of interest to declare.