cAMP-PKA Signaling Plays an Important Role in Augmented Neutrophil Adhesion and Chemotaxis in Sickle Cell Disease.
Authors: Franco-Penteado Carla , Canalli Andreia A. , Ferreira Renata P. , Saad Sara T.O. , Costa Fernando F. , Conran Nicola 
Published: April 2020

Abstract
A chronic inflammatory state is now thought to underlie much of the pathophysiology of sickle cell disease (SCD). Increased leukocyte numbers are associated with increased morbidity and mortality in SCD and the adhesion of leukocytes to the vascular endothelium and sickle red cells may play an initiating role in the vaso-occlusive process. We sought to better understand the signaling events that lead to alterations in SCD neutrophil adhesive and chemotactic abilities. Neutrophils were isolated from the peripheral blood of healthy controls and SCD individuals in steady state by separation over a ficoll-paque gradient. Following washing and lysis of contaminating red cells, adhesion to fibronectin (FN) and chemotaxis of control and SCD neutrophils (4×106 cells/ml in RMPI medium) were assessed using static adhesion assays and a 96-well chemotaxis chamber assay (ChemoTX, Neuroprobe). Cell adhesion to FN (20mg/ml; 30 min, 37°C, 5% CO2) or IL-8-induced migration (2h, 37°C, 5% CO2) was calculated using a standard curve of the original cell suspension and expressed as the percentage of cells adhered or the number of cells migrated. As previously shown, SCD neutrophils demonstrate a greater ability to adhere to FN-coated plates than control individual leukocytes (13.1±1.48 % neutrophils adhered compared to 5.0±0.6 %; n=11; P&lt;0.001 Mann-Whitney test). Interestingly, SCD neutrophils demonstrate significantly higher levels of the secondary messenger, cAMP, than control neutrophils (4.76±0.38 pMol/106 cells compared to 1.91±0.34 pMol/106 cells; n≥12; P&lt;0.001). When neutrophils were incubated on FN-coated plates in the presence of the cAMP-dependent kinase (PKA) inhibitor, KT5720 (3μM), increased SCD neutrophil adhesion was significantly reversed to levels approaching those of control neutrophils (7.73±1.3 %; n=11; P=0.001). In contrast, control neutrophil adhesion to FN was not significantly affected by the PKA inhibitor (P&gt;0.05). Furthermore, activation of primed SCD neutrophils by the chemokine, IL-8 (500ng/ml), resulted in a significant increase in SCD neutrophil adhesion to FN (19.3±2.2 %; P=0.02; n=7) with a concomitant increase in intracellular cAMP levels (335.8±17.1 % increase, P&lt;0.05, n=3) that could also be reverted by co-incubation with the PKA inhibitor, KT5720 (11.8 ± 0.8 %; P=0.02; n=7). Interestingly, in vitro chemotaxis of SCD neutrophils in response to a stimulus of IL-8 (100 ng/ml) was found to be significantly increased when compared to control neutrophil IL-8-stimulated chemotaxis (14.3±1.1 x105 cells/ml migrated compared to 8.8±0.8x105 cells/ml; P=0.005; n=8). Once again, KT5720 (3 μM, pre-incubation for 15 min, 37°C), was able to significantly reverse (8.5±1.1 x105/ml; P=0.03; n=6) this enhanced IL-8-dependent SCD neutrophil chemotaxis. Results suggest that an up-regulated PKA-dependent pathway may play an important role in the altered adhesive and chemotactic properties of SCD neutrophils and their further activation by cytokines/chemokines such as IL-8, found in increased levels in the serum of SCD patients. Thus, upregulation of the cAMP-PKA pathway in SCD neutrophils may have important consequences for the chronic inflammatory state in SCD patients and may characterize a novel target for the control of altered neutrophil function in SCD.