Heterotypic interactions of neutrophils in sickle cell anemia patients

Sickle cell anemia (SCA) is a hereditary hemoglobinopathy that results from a mutation in the beta globin gene. This mutation leads to the formation of an abnormal hemoglobin (Hb), known as HbS. Vaso-occlusion is the major acute complication and the major cause of morbidity in SCA; it comprises a multicellular process that appears to be initiated by the adhesion of red blood cells (RBCs) and leukocytes to the activated endothelium, causing vascular obstruction and tissue ischemia. The adhesion of sickle RBCs to the endothelium contributes to decrease blood flow; however, leukocytes also seem to play a key role in this process as these cells are bigger, less flexible and their recruitment to the microvasculature and subsequent interaction with circulating blood cells promotes reduced blood flow in vessels. Research into the dynamics of heterocellular interactions is a potentially attractive area of research, since such data may improve our understanding of the mechanisms involved in vascular inflammation and contribute to the development of new therapeutic targets in diseases such as SCA. The aim of this project was to study the heterotypic interactions of neutrophils with platelets, red cells and endothelial cells in cells from SCA individuals and the possible adhesion molecules involved in these interactions, using microfluidic techniques (cell adhesion flow in biochips with parallel channels), imaging flow cytometry (simultaneous imagery of cells acquired by flow cytometry) and the development of a biochip for the study of vaso-occlusive processes (for use in conjunction with the microfluidic platform). Peripheral blood from healthy individuals (controls) and sickle cell anemia patients (with or without hydroxyurea therapy - HU) were collected and the experiments were performed. RBCs from SCA patients without HU were observed to adhere more, under flow, to laminin-coated biochips and they interact more with autologous neutrophils previously adhered to an activated endothelium. When evaluating the participation of the two main adhesion molecules expressed on activated endothelium, E- selectin and ICAM-1, in promoting interactions of RBCs to adherent neutrophils, we found that the duration of these heterotypic interactions for cells from SCA patients was longer in the presence of E-selectin ligand. Neutrophils from SCA patients also circulate aggregated to platelets or RBCs in the peripheral blood significantly more than the neutrophils of healthy individuals. Reticulocytes were the main RBC type involved in these neutrophil-RBC aggregates and the levels of fetal hemoglobin were inversely correlated to the percentage of the neutrophil- reticulocyte aggregates found in the peripheral blood of these patients. Platelets demonstrated a prominent role in the formation of the circulating neutrophil-RBCs aggregates, and function-blocking antibodies or inhibitors indicated that the adhesion molecules P-selectin, on platelets, Mac-1, on neutrophils, VLA-4, on reticulocytes, and ICAM-4, on mature erythrocytes, may be responsible for the interactions of neutrophils with these cells. Neutrophils from SCA patients also demonstrated a significant capacity to obstruct microchannels with diameters of between 25 and 40?m, of the biochip developed by us to study vaso-occlusive processes, compared to the cells from control subjects. When mixed suspensions of neutrophils and autologous RBCs were applied to the chips, we observed an even greater obstruction of these microchannels. In summary, we conclude that SCA neutrophils interact significantly more with red cells when adhered to endothelial cells, circulate aggregated to erythrocytes in the peripheral blood of these patients, and present a greater capacity to obstruct microchannels that mimic small venules in vitro. HU therapy appears to affect only neutrophil interactions in the presence of the vascular endothelium. Therapies that target molecules such Mac-1 and P-selectin in sickle cell disease seem to be promising strategies (AU)