[Cytometry and cell sorting].

We have recently shown that IL-10 fails to trigger Stat3 and Stat1 tyrosine phosphorylation in freshly isolated human neutrophils. In this study, we report that IL-10 can nonetheless induce Stat3 tyrosine phosphorylation and the binding of Stat1 and Stat3 to the IFN-gamma response region or the high-affinity synthetic derivative of the c-sis-inducible element in neutrophils that have been cultured for at least 3 h with LPS. Similarly, the ability of IL-10 to up-regulate suppressor of cytokine signaling (SOCS)-3 mRNA was dramatically enhanced in cultured neutrophils and, as a result, translated into the SOCS-3 protein. Since neutrophils' acquisition of responsiveness to IL-10 required de novo protein synthesis, we assessed whether expression of IL-10R1 or IL-10R2 was modulated in cultured neutrophils. We detected constitutive IL-10R1 mRNA and protein expression in circulating neutrophils, at levels which were much lower than those observed in autologous monocytes or lymphocytes. In contrast, IL-10R2 expression was comparable in both cell types. However, IL-10R1 (but not IL-10R2) mRNA and protein expression was substantially increased in neutrophils stimulated by LPS. The ability of IL-10 to activate Stat3 tyrosine phosphorylation and SOCS-3 synthesis and to regulate IL-1 receptor antagonist and macrophage-inflammatory protein 1beta release in LPS-treated neutrophils correlated with this increased IL-10R1 expression, and was abolished by neutralizing anti-IL-10R1 and anti-IL-10R2 Abs. Our results demonstrate that the capacity of neutrophils to respond to IL-10, as assessed by Stat3 tyrosine phosphorylation, SOCS-3 expression, and modulation of cytokine production, is very dependent on the level of expression of IL-10R1.

Cell Separation↗

PubMed2001

Experimental design methodology applied to the study of channel dimensions on the elution of red blood cells in gravitational field flow fractionation.

Field flow fractionation (FFF) separation techniques have gained considerable success with micron-sized species. Living red blood cells (RBCs) of any origin have emerged as ideal models for cell separation development. Their elution mode is now described as "Lift-Hyperlayer". Certain separator dimension parameters are known to play a key role in the separation and band spreading process. Systematic studies of channel dimensions effects on RBC retention, band spreading, peak capacity and on a novel parameter described as "Particle Selectivity" were set up by means of a two-level factorial experimental design. From experimental results and statistical calculations it is confirmed that channel thickness plays a major role in retention ratio, peak variance, peak capacity and particle selectivity. Channel breadth strongly influences plate height, with lower impact on peak capacity and particle selectivity. Retention ratio, peak variance and peak capacity observed results are modulated by second-order interactions between channel dimensions. Preliminary rules for channel configurations are therefore set up and depend on separation goals. It is shown that a very polydisperse population is best disentangled in a thin and narrow channel whatever its length. If a mixture of many different micron-sized species is considered (each of limited polydispersities); a thick and broad channel should be preferred, with length modulating peak capacity to disentangle this polymodal mixture.

Cell Separation↗