Human Sickle Cell Disease Increases Numbers and Activation Of Peripheral Blood Myeloid Dendritic Cells, Monocytes, and Neutrophils

Sickle cell disease (SCD) is characterized by widespread vaso-occlusion in venules that is initiated by the polymerization of deoxy-hemoglobin in sickle RBCs and exacerbated by leukocyte activation and endothelial injury. In the course of the disease, episodic flare-ups result in painful vaso-occlusive crises (pVOC) and acute chest syndrome that over time result in chronic tissue injury. We have previously shown that pVOC in SCD is associated with activation of a small subset of CD1d restricted T lymphocytes known as invariant NKT (iNKT) cells that release large amounts of pro-inflammatory cytokines and propagate an inflammatory cascade. Antigen presenting cells (APCs) stimulate the activation of iNKT cells by presenting lipid antigens on CD1d and by releasing co-activating cytokines such as IL-12 and IL-18. Cytokines released by activated iNKT cells such as IFN-γ further propagate inflammation by stimulating chemotaxis of and activation of additonal leukocytes. In mice, the cytokines that are released by activated iNKT cells trans-activate monocytes and neutrophils. In this study we investigated for the first time the effect of pVOC in patients on the activation of myeloid (CD11c⁺/CD123^-) dendritic cells (DCs) (CD45⁺/Lin1^-/HLA-DR⁺), monocytes (CD3^-/CD19^-/CD15^-/CD66b^-/CD14⁺), and neutrophils (CD3^-/CD19^-/CD15⁺/CD66b⁺). Our findings indicate that like iNKT cells, myeloid DCs, monocytes, and neutrophils are increased in peripheral blood of SCD subjects relative to non-SCD controls. Plasmacytoid DCs (CD11c^-/CD123⁺) were little affected. This increase in cell numbers is enhanced during pVOC except for myeloid DCs which decrease, possibly due to extravasation out of the blood stream. In addition, CD1d⁺ myeloid DCs from SCD individuals express higher levels of the activation marker, CD86, and this is further increased during pVOC. Similarly, neutrophils and monocyte subsets including classical monocytes (CD14⁺/CD16^-) and patrolling monocytes (CD14^dim/CD16⁺) express higher levels of activated adhesion molecules, LFA-1 (detected with KIM127 antibody) and Mac-1 (detected with CBRM1/5 antibody that recognizes active CD11b), in SCD subjects during pVOC as compared to controls and steady-state SCD patients. Taken together, these findings strongly suggest that the severity of vaso-occlusion during the clinical course of SCD correlates with an increased pro-inflammatory state reflected by increased circulating activated leukocytes. These findings are also consistent with previous murine studies showing that iNKT cells are necessary, but not sufficient for initiating and amplification of tissue inflammation and damage. The activation of CD1d+ APCs during pVOC is likely to facilitate iNKT cell activation due to the expression of co-stimulatory molecules (CD86), inflammatory cytokines, and possibly lipid antigens. Moreover, the activation of adhesion molecules on monocytes and neutrophils may enhance their propensity to contribute to vaso-occlusion.