Hydroxyethyl Starch 130/0.4 Binds to Neutrophils Impairing Their Chemotaxis through a Mac-1 Dependent Interaction

Abstract

Several studies showed that hydroxyethyl starch (HES), a synthetic colloid used in volume replacement therapies, interferes with leukocyte-endothelium interactions. Although still unclear, the mechanism seems to involve the inhibition of neutrophils' integrin. With the aim to provide direct evidence of the binding of HES to neutrophils and to investigate the influence of HES on neutrophil chemotaxis, we isolated and treated the cells with different concentrations of fluorescein-conjugated HES (HES-FITC), with or without different stimuli (N-Formylmethionine-leucyl-phenylalanine, fMLP, or IL-8). HES internalization was evaluated by trypan blue quenching and ammonium chloride treatment. Chemotaxis was evaluated by under-agarose assay after pretreatment of the cells with HES or a balanced saline solution. The integrin interacting with HES was identified by using specific blocking antibodies. Our results showed that HES-FITC binds to the plasma membrane of neutrophils without being internalized. Additionally, the cell-associated fluorescence increased after stimulation of neutrophils with fMLP (p < 0.01) but not IL-8. HES treatment impaired the chemotaxis only towards fMLP, event mainly ascribed to the inhibition of CD-11b (Mac-1 integrin) activity. Therefore, the observed effect mediated by HES should be taken into account during volume replacement therapies. Thus, HES treatment could be advantageous in clinical conditions where a low activation/recruitment of neutrophils may be beneficial, but may be harmful when unimpaired immune functions are mandatory.

Keywords: Chemotaxis; Hydroxyethyl Starch; IL-8; Neutrophil; fMLP; integrin; volume replacement solutions.

Figure 1

Association of HES to the outer plasma membrane of neutrophils. (A) Neutrophils were treated with different concentrations of FITC-labeled HES, washed and the resulting fluorescence read with a microplate fluorimeter. There was an increase in fluorescence with increasing concentrations of HES-FITC (n = 3). (B) After the treatment with HES-FITC and washing steps, neutrophils were incubated with PBS or NH₄Cl in order to rule out a possible internalization of HES into phagolysosomes. No significant difference in the fluorescence of the cells treated with NH₄Cl compared to the control was observed, suggesting that HES was bound to the outer plasma membrane and not internalized (n = 3). The data are presented as mean ± SD ** p < 0.01.

Figure 2

Increase in the binding of hydroxyethyl starch (HES) after neutrophils stimulation. Neutrophils were either activated with fMLP, IL-8 or not stimulated and then incubated with HES-FITC. After washing steps, the fluorescence was read with a microplate fluorimeter and the values were reported as percentage of binding with respect to the not stimulated condition. There was a significant increase in the binding of HES after fMLP stimulation but not after IL-8 treatment. The data represents the mean ± SD of five independent experiments. ** p < 0.01.

Figure 3

Migration of neutrophils not treated (control) or treated (+ HES) with hydroxyethyl starch in response to IL-8. Low magnification time-lapse images were obtained and processed using cell-tracking algorithms. (A) Experimental setup. (B) Detailed migration trajectories of representative cells (n = 100) from control and HES-treated cells; individual tracks were transposed so that each had its start at the origin. (C) Comparison of forward migration index (FMI), parallel to the gradient and calculated as the ratio between the x coordinate of the cell’s end point of migration and the total distance accumulated), directionality (representing a measurement of the directness of cell trajectories, the ratio of the Euclidian distance and the total accumulated distance of a cell), and velocity (displacement/time). None of the values were different between the control and the cells treated with HES, suggesting that hydroxyethyl starch did not impair the migration of cells in response to IL-8. The results are the mean ± SD of three independent experiments.

Figure 4

Migration of neutrophils not treated (control) or treated (+ HES) with Hydroxyethyl starch in response to fMLP. Low magnification time-lapse images were obtained and processed using cell-tracking algorithms. (A) Experimental setup. (B) Detailed migration trajectories of representative cells from control (n = 62) and HES-treated (n = 17) cells; individual tracks were transposed so that each had its start at the origin. (C) Comparison of forward migration index, directionality, and velocity. As can be seen, the cells pre-treated with HES showed a decreased FMI, directionality and velocity compared to the control. The results are the mean ± SD of three independent experiments. ** p < 0.01, *** p < 0.001

Figure 5

Blocking of integrin subunits with specific antibodies. The cells were incubated with blocking antibodies specific for either CD11a (black bar), CD11b (dashed bar), CD18 (gray bar) or isotypic control (open bar) and treated with HES-FITC. The fluorescence was recorded after washing steps and the data were represented as percentage of fluorescence with respect to the control. We observed that the blocking of CD11b or CD18 subunits, but not CD11a, prevented the binding of HES (decrease in the membrane-associated fluorescence) to neutrophils. The data represent the mean ± SD of five independent experiments. * p < 0.05; ** p < 0.01.

Figure 6

Static adhesion assay performed on different substrates. Cells were fluorescently labeled with calcein and then subjected to adhesion on different substrates. (A) When fibrinogen was used as a substrate for adhesion, HES pre-treatment strikingly decreased (almost 50%) the adhesion of cells. (B) The adhesion of neutrophils on JAM-1 was not impaired by HES, suggesting a Mac-1 specific effect. The presented results are the mean ± SD of four independent experiments. ** p < 0.01.

Figure 7

Evaluation of integrin activation by HES. (A) HES-mediated outside-in integrin activation was evaluated by determining total phosphorylation of tyrosine on intracellular proteins through Western blot (only a representative blot is showed in the picture). HES caused a significant increase in total phosphorylation compared to the control. (B) Evaluation of Akt activation by Western blot. HES treatment significantly increased the phosphorylation of Akt the activation of this pathway. (C) Evaluation of p38 activation by Western blot. As reported, neither HES nor ICAM caused the activation of the p38/MAPK pathway. In the blots, lane C represents the control. The presented results are the mean ± SD of three independent experiments. * p < 0.05.