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

Abstract

BackgroundLeukocyte-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. MethodsThis 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. ResultsRapid 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. ConclusionsThe 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s13054-015-1157-5) contains supplementary material, which is available to authorized users.

Single cell rheometry with a microfluidic constriction: Quantitative control of friction and fluid leaks between cell and channel walls

“…Conversely, the double constriction device allows an accurate comparison of cell properties in a wide range of stiffness, which has allowed us to investigate pathologies involving the plugging of lung microvasculature by stiffened leukocytes. 56 In the present work, we demonstrate how this device can also be used to infer quantitative rheological analysis on single cells.…”

Neutrophil-Mediating Lung Diseases and Integrin α subfamily, α4 and α9

“…Not only in animal model, but also human septic cases showed up-regulation of integrin α4β1on neutrophils [9,10] . On the other hand, increased expression of integrin α4β1on neutrophils was not recognized in early stage of acute respiratory distress syndrome (ARDS) cases in another paper [11] . They discussed that increased expression level of adhesion molecule might be delay, several hours later.…”

The mechanics of myeloid cells

“…Neutrophil size increases in trauma patients prior to organ dysfunction [Hesselink et al., ] and neutrophil deformability decreases in patients with chronic renal failure [Skoutelis et al., ]. Treatment of neutrophils and monocytes with serum from patients with acute respiratory distress syndrome induces rapid cellular stiffening [Preira et al., ]. In line with this observation, RT‐DC showed that patients with acute lung injury (ALI) and patients hospitalised with viral respiratory tract infections (RTI) have larger and more deformable neutrophils in their blood than healthy volunteers [Toepfner et al., ].…”