Red Cell Exchange Decreases Sickle RBC-Induced Release of MPO from Neutrophils

Introduction: Neutrophil activation has long been recognized to contribute to sickle cell disease (SCD) pathophysiology. In prior studies, we have shown that patients with SCD have activated neutrophils with enhanced degranulation responses as measured by matrix metalloproteinase-9 (MMP9) release, representative of tertiary granules. Furthermore, both simple transfusion and red cell exchange (RCE) decrease neutrophil activation and MMP9 release (Lee et al, Transfusion 2024), while exposure of normal neutrophils to sickle red cells (SS RBCs) promotes neutrophil activation, inducing both increased neutrophil adhesion to endothelium as well as MMP9 release from tertiary granules (Lee et al. Brit J Haem 2024). It is unknown if these observations extend to other neutrophil granule populations.

Methods/Results: We first determined if SS RBCs induce release of neutrophil primary granules. With IRB approval, healthy neutrophils were incubated with SS RBCs or AA RBCs (n=6/cohort), and myeloperoxidase (MPO) release was measured by ELISA (R&D Systems). SS RBCs from 5 of the 6 SCD patients induced significantly more MPO release from neutrophils isolated from ABO-compatible healthy donors as compared to AA RBCs incubated with the same healthy neutrophils (average ± SEM MPO release with AA RBC 494.6 ± 68.5 pg/mL vs. SS RBC 859.5 ± 243.2 pg/mL, p=ns; average ± SEM increase in MPO release with SS RBC 60.8 ± 26.7%) To determine if RCE modulates this SS RBC-induced release of neutrophil primary granules, whole blood from SCD patients at steady-state who were receiving non-emergent, outpatient RCE (n=25) was obtained before and after RCE. After incubation with buffer or one of several agonists, MPO released into supernatant was measured by ELISA. RCE significantly decreased neutrophil release of MPO after exposure to buffer alone (buffer pre-RCE 1442 ± 1242 pg/mL vs. post-RCE 678 ± 408 pg/mL, p=0.003). RCE also consistently reduced neutrophil release of MPO after exposure to each of several agonists: phorbol 12-myristate 13-acetate (PMA 500 nM), N-Formyl-Met-Leu-Phe (fMLF 1 uM), or lipopolysaccharide (LPS 100 ng/mL). Reduction in neutrophil degranulation and MPO release was: PMA pre-RCE 3102 ± 1237 pg/mL vs. post-RCE 2193 ± 1019 pg/mL, p=0.001; fMLF pre-RCE 5236 ± 1496 pg/mL vs. post-RCE 3263 ± 1367 pg/mL, p<0.0001; and LPS pre-RCE 3760 ± 2051 vs. post-RCE 1333 ± 680.3 pg/mL; p<0.0001. These results are consistent with a reduction in neutrophil priming prior to exposure to agonist.

Conclusions: Taken together, these studies demonstrate that SS RBCs directly induce multiple events associated with neutrophil activation, resulting in release of neutrophil primary granules containing MPO in addition to the previously described release of MMP9 from tertiary granules. Moreover, we show that RCE significantly modulates neutrophil hyper-responsiveness, resulting in attenuated primary granule release at baseline (neutrophils exposed to buffer only) as well as in response to a range of agonists, including bacterial ligands. These results add to the growing recognition that SS RBCs contribute to neutrophil activation, and that transfusion modulates neutrophil activation in SCD. At present, standard practice is to calibrate RCE to achieve a pre-specified reduction in HbS levels after exchange transfusion. However, the effects of RCE on neutrophil activation offer another pathophysiologically relevant marker of RCE efficacy that could help elucidate how RCE exerts its benefits, as well as how it might fail to do so in some instances. Further exploration of the effects of RCE on neutrophil activation in both steady state and in patients with acute SCD complications treated by RCE is warranted.

Telen:GBT/Pfizer: Research Funding; Novo Nordisk: Research Funding; CSL Behring: Research Funding. Lee:Gilero: Research Funding; Zymeron: Research Funding; Novartis: Research Funding; Sanofi: Research Funding.