The leukocyte-stiffening property of plasma in early acute respiratory distress syndrome (ARDS) revealed by a microfluidic single-cell study: the role of cytokines and protection with antibodies

Pascal Preira, Jean-Marie Forel, Philippe Robert, Paulin Nègre, Martine Biarnes-Pelicot, Francois Xeridat, Pierre Bongrand, Laurent Papazian, Olivier Theodoly

Leukocyte-mediated pulmonary inflammation is a key pathophysiological mechanism involved in acute respiratory distress syndrome (ARDS). Massive sequestration of leukocytes in the pulmonary microvasculature is a major triggering event of the syndrome. We therefore investigated the potential role of leukocyte stiffness and adhesiveness in the sequestration of leukocytes in microvessels.  This study was based on in vitro microfluidic assays using patient sera. Cell stiffness was assessed by measuring the entry time (ET) of a single cell into a microchannel with a 6 × 9-μm cross-section under a constant pressure drop (ΔP = 160 Pa). Primary neutrophils and monocytes, as well as the monocytic THP-1 cell line, were used. Cellular adhesiveness to human umbilical vein endothelial cells was examined using the laminar flow chamber method. We compared the properties of cells incubated with the sera of healthy volunteers (n = 5), patients presenting with acute cardiogenic pulmonary edema (ACPE; n = 6), and patients with ARDS (n = 22), of whom 13 were classified as having moderate to severe disease and the remaining 9 as having mild disease.  Rapid and strong stiffening of primary neutrophils and monocytes was induced within 30 minutes (mean ET >50 seconds) by sera from the ARDS group compared with both the healthy subjects and the ACPE groups (mean ET <1 second) (p < 0.05). Systematic measurements with the THP-1 cell line allowed for the establishment of a strong correlation between stiffening and the severity of respiratory status (mean ET 0.82 ± 0.08 seconds for healthy subjects, 1.6 ± 1.0 seconds for ACPE groups, 10.5 ± 6.1 seconds for mild ARDS, and 20.0 ± 8.1 seconds for moderate to severe ARDS; p < 0.05). Stiffening correlated with the cytokines interleukin IL-1β, IL-8, tumor necrosis factor TNF-α, and IL-10 but not with interferon-γ, transforming growth factor-β, IL-6, or IL-17. Strong stiffening was induced by IL-1β, IL-8, and TNF-α but not by IL-10, and incubations with sera and blocking antibodies against IL-1β, IL-8, or TNF-α significantly diminished the stiffening effect of serum. In contrast, the measurements of integrin expression (CD11b, CD11a, CD18, CD49d) and leukocyte-endothelium adhesion showed a weak and slow response after incubation with the sera of patients with ARDS (several hours), suggesting a lesser role of leukocyte adhesiveness compared with leukocyte stiffness in early ARDS.  The leukocyte stiffening induced by cytokines in the sera of patients might play a role in the sequestration of leukocytes in the lung capillary beds during early ARDS. The inhibition of leukocyte stiffening with blocking antibodies might inspire future therapeutic strategies.