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. 2023 Sep 1;69(9):841-848.
doi: 10.1097/MAT.0000000000001985. Epub 2023 May 9.

Neutrophil Structural and Functional Alterations After High Mechanical Shear Stress Exposure

Affiliations

Neutrophil Structural and Functional Alterations After High Mechanical Shear Stress Exposure

Katherin Arias et al. ASAIO J. .

Abstract

Patients on mechanical circulatory support are prone to infections, increasing morbidity and mortality. These circulatory support devices generate high mechanical shear stress (HMSS) that can causes trauma to blood. When leukocytes become damaged, their immune response function may be impaired or weakened, leading to increased infection vulnerability. This study examined neutrophil structural and functional alterations after exposure to 75, 125, and 175 Pa HMSS for 1 second. Human blood was exposed to three levels of HMSS using a blood shearing device. Neutrophil morphological alteration was characterized by examining blood smears. Flow cytometry assays were used to analyze expression levels of CD62L and CD162 receptors, activation level (CD11b), and aggregation (platelet-neutrophil aggregates). Neutrophil phagocytosis and rolling were examined via functional assays. The results show neutrophil structure (morphology and surface receptors) and function (activation, aggregation, phagocytosis, rolling) were significantly altered after HMSS exposure. These alterations include cell membrane damage, loss of surface receptors (CD62L and CD162), initiation of activation and aggregation, upregulation of phagocytic ability and increased rolling speed. The alterations were the most severe after 175 Pa exposure. HMSS caused damage and activation of neutrophils, potentially impairing normal neutrophil function, leading to weakened immune defense and increasing a patient's vulnerability to infections.

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Conflict of interest statement

Disclosure: The authors have no conflicts of interest to report.

Figures

Figure 1:
Figure 1:
Overview of experimental set up using a Couette-type blood-shearing device (Hemolyzer).
Figure 2:
Figure 2:
(A) Representative images of neutrophil morphology before and after shear stress exposure of 75, 125, and 175 Pa for 1 sec. (B) The percentage of cell damaged was analyzed based on membrane integrity (mean ± SE), n=4. **P< 0.05.
Figure 3:
Figure 3:
(A) Surface expression of CD62L on CD66b+ neutrophils before and after shear stress exposure for 1 second at 75, 125, and 175 Pa. (B) Surface expression of CD162 on CD66b+ neutrophils before and after shear stress exposure of 75, 125, and 175 Pa for 1 sec. **P< 0.05.
Figure 4:
Figure 4:
(A) Flow cytometry gating strategy for CD11b expression on CD66b+ neutrophils and representative plots from one experiment for base and 175 Pa samples. (B) Surface expression of CD11b on CD66b+ neutrophils before and after shear stress exposure of 75, 125, and 175 Pa for 1 sec. ** P< 0.05. (C) Representative FlowSight intensity images showing bright field and fluorescent CD11b+ cell images (20X) from Amnis image flow cytometry.
Figure 5:
Figure 5:
(A) Percentage of CD66b+ neutrophils that expressed CD41a (platelet marker) before and after shear stress exposure of 75, 125, and 175 Pa for 1 sec. ** P< 0.05. (B) Representative neutrophil-platelet aggregate images for 75, 125, and 175 Pa samples showing bright field and fluorescent CD66b+ and CD41a+ cell images (20X) from Amnis image flow cytometry.
Figure 6:
Figure 6:
(A) Flow cytometry gating strategy for phagocytosis of DyLight 633 labeled latex beads on CD66b+ neutrophils and representative plots from one experiment for base and 175 samples. (B) Percentage of CD66b+ neutrophil that underwent phagocytosis before and after shear stress exposure of 75, 125, and 175 Pa for 1 sec (mean ± SE). **P < 0.05.
Figure 7:
Figure 7:
(A) Representative video frames of a leukocyte rolling track at various timepoints. (B) Leukocyte rolling speed (μm/s) on P-selectin before and after shear stress exposure of 75, 125, and 175 Pa for 1 sec (mean ± SE). **P < 0.05.

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