Slow flow of passive neutrophils and sequestered nucleus into micropipette.

In the present study, the role of the nucleus and its contribution to the deformability of the passive neutrophils was investigated. To determine the rheological properties of the nucleus and of the neutrophil itself, deformation tests on single neutrophil and sequestered nucleus have been performed by micropipette under low aspiration pressure (80 Pa = 2-3 Pcr). The stiffness of the nucleus was found to be larger than that of the neutrophil, and its viscosity was found almost ten-fold higher. A subpopulation of neutrophils (Sub-A) showed two phases of deformation, a first rapid phase and a second phase with a constant deformation rate up to their full entrance, with an apparent viscosity mu app-second-Phase(N Sub-A) = 286 +/- 123 Pa x s, calculated by the liquid drop model. Another subpopulation (Sub-B) of the tested neutrophils displayed three deformation phases: a first one reflecting the rapid entry of cell into the micropipette, a second with constant deformation rate, and a third phase, with a slower, also constant, deformation rate were recorded. The corresponding apparent viscosities were found as mu app-second-Phase(N Sub-B) = 341 +/- 94 Pa x s and mu app-third-Phase(N Sub-B) = 1651 +/- 734 Pa x s. The apparent viscosity values of the neutrophilic nucleus, mu app (N nucl) = 2468 +/- 1345 Pa x s and of the whole neutrophil calculated in the third phase of deformation, mu app-third-Phase(N Sub-B) = 1651 +/- 734 Pa.s were comparable. These results support our hypothesis that the nucleus plays a significant role in the mechanical and rheological behavior of the neutrophil, especially when it has to pass through openings much smaller than its size.

Polymorphonuclear leukocyte rigidity is defective in patients with chronic renal failure.

BACKGROUND: The purpose of the study was to investigate the rigidity of polymorphonuclear leukocytes (PMNs) in non-dialysed chronic renal failure (CRF) and haemodialysis (HD) patients. METHODS: PMN rigidity as well as tumour necrosis factor alpha (TNF-alpha) and interleukin 1beta (IL-1beta) plasma levels were assessed in 10 early-stage CRF, 10 late-stage non-HD, and 10 HD patients, before and during dialysis. In HD patients both cellulose acetate and polysulphone membranes were used. Ten healthy subjects served as controls. Rigidity was tested by counting the deformability in morphologically passive PMNs by the micropipette method. Cytokine levels were measured by enzyme-linked immunosorbent assay. RESULTS: PMN rigidity was significantly increased in end-stage CRF patients regardless of HD but not in early-stage CRF. In HD patients PMN rigidity increased significantly 60 min after initiation of HD. There was an increase of TNF-alpha and IL-1beta levels in end-stage non-HD and HD patients and a further increase at 60 min after initiation of HD. The percentage of morphologically activated PMNs was increased only during dialysis. The nature of the HD membrane had no influence on rigidity, PMN activation, or cytokine production. CONCLUSIONS: The results indicate that PMN rigidity is defective in end-stage chronic CRF patients and is further increased 60 min after initiation of HD, regardless of the nature of the HD membrane used. PMN activation, increased TNF-alpha and IL-1beta levels, or a direct PMN impairment may cause the observed cell rigidity.

Effect of cytokines and colony-stimulating factors on passive polymorphonuclear leukocyte deformability in vitro.

Cytokines are potent polymorphonuclear leukocyte (PMN) activators and can decrease their deformability. We evaluated passive PMN deformability using the micropipette method after incubation with different concentrations of lipopolysaccharide (LPS), interleukins (IL-) 1, 6, 8 and 10, tumour necrosis factor (TNF), granulocyte (G) and granulocyte-macrophage (GM) colony-stimulating factors (CSF). TNF, IL-1, G-CSF, GM-CSF and, to a lesser degree, IL-6 significantly and in a dose-dependent fashion decrease PMN deformability. LPS had no direct effect on PMN deformability. When cytokines at concentrations with no effect on deformability were combined they increased PMN rigidity. The findings suggest that several cytokines and CSF impair directly, and not by activation alone, PMN deformability.

Neutrophil deformability in patients with sepsis, septic shock, and adult respiratory distress syndrome.

OBJECTIVE: To investigate the deformability of morphologically active and passive neutrophils in patients with sepsis (SP), septic shock (SS), and adult respiratory distress syndrome (ARDS). DESIGN: Prospective, observational study. SETTING: A university hospital intensive care unit and research laboratory. PATIENTS: Six patients with sepsis, six patients with septic shock, and six patients with ARDS. Eight healthy volunteers and eight ventilated but noninfected patients served as controls. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Morphologically passive and active neutrophil deformability as defined by the micropipette method was significantly decreased in patients with SP, SS, and ARDS associated with sepsis as compared with both control groups. Neutrophils from SS and ARDS patients were significantly more rigid as compared with neutrophils from SP patients but they did not differ from each other. The percentage of activated neutrophils was significantly higher in SP, SS, and ARDS patients. Increased passive neutrophil rigidity was significantly attenuated after coincubation with cytochalasin D. Tumor necrosis factor-alpha and interleukin-1beta serum levels were significantly higher in SP, SS, and ARDS patients. CONCLUSIONS: The entire neutrophil population is less deformable in SP, SS, and ARDS patients. The decreased deformability of passive neutrophils suggests that a direct mechanism involving actin polymerization, distinct from cell activation, is involved. These observations may be important in the mechanism of impaired vascular flow in patients with sepsis.

Deformability and filterability of white blood cell subpopulations. Evaluation of these parameters in the cell line HL-60 and in type II diabetes mellitus.

The rheological properties of human leukocytes (WBCs) have been studied using the micropipette aspiration and the filtration technique. Partial micropipette (i.d. 2.8-4.5 microm) aspiration of individual leukocytes under constant aspiration pressure of 8 mm H20 and measurement of the aspirated length as a function of time (creep experiments) according to the Evans model have been carried out and the apparent viscosity mu(app) was estimated. In the filtration experiments, using the Hemorheometer, the Index Rigidity of Leukocytes, ILR, was also estimated. The apparent viscosity mu(app) of normal PMN and MNC was significantly different p < 0.05), while the LYM and PMN had no statistical difference (p < 0.5). The leukocytes of the cell line HL-60 were more rigid than the normal PMN (p < 0.01), while the PMN from patients with type II diabetes mellitus were more rigid than the normal PMN (p < 0.005). The results of IRL showed similar differences among all of the leukocyte subpopulations. Comparison of these findings suggests a possible relationship between ILR and mu(app) which in this case is: ILR = 598 + 0.54 mu(app) (r = 0.986, p-value 0.0003).