Heterogeneity in the circulating neutrophil pool: studies on subpopulations separated by continuous flow electrophoresis

Abstract

Isolated blood neutrophils from healthy individuals have been separated by continuous flow electrophoresis (CFE) as a Gaussian-shaped profile extending over 12-15 fractions, on the basis of differences in cell surface electrical charge. The fractions were pooled into three or four subpopulations; the mean electrophoretic mobilities of the least and most electronegative cells were 0.96 and 1.22 microns/sec/V/cm, respectively. Each pool of neutrophils was analyzed for functional and biochemical differences. Expression of respiratory burst in terms of the rate of superoxide production by the least and most electronegative cells to fixed concentrations of N-formyl-methionyl-leucyl-phenylalanine (fMLP, 10(-7) M) or phorbol myristate acetate (PMA, 1 microgram/ml) revealed that the least electronegative cells generated superoxide anion (O2-) at approximately twice the rate of the most electronegative cells. However, when lower concentrations of fMLP were used (1-5 x 10(-9) M), the most electronegative cells were most active. The least electronegative cells were also the most active in terms of phagocytosis and chemotaxis. In accordance with these differences in motile function, the basal F-actin content of the least electronegative cell pool was greater than the F-actin levels found in the most electronegative cells and remained so upon stimulation with fMLP. Such neutrophil heterogeneity as detected by CFE may be important in selective margination and recruitment of cells to inflammatory foci and sites of infection and may in part represent subsets of cells within the circulation that are primed in vivo to respond to inflammatory stimuli.

Differences in oxidative response of subpopulations of neutrophils from healthy subjects and patients with rheumatoid arthritis.

“…Priming of these cells with PAF did not enhance their generation of 02production by FMLP, which was in contrast to the neutrophils with a high net negative surface charge, which exhibited significant increases in responsiveness following PAF priming. These findings add support to the concept that there is functional and metabolic heterogeneity within neutrophils,917 and that the magnitude techniques (continuous flow electrophoresis and partitioning in two phase aqueous polymer systems), our earlier studies showed that blood neutrophils are heterogeneous with respect to cell surface membrane electrokinetic properties and functional responsiveness.3 14 Our previous work also demonstrated that neutrophils of a low electrophoretic mobility were increased in the blood and particularly in the synovial fluid of RA patients,9 1 and that immune complexes reduced the surface charge of normal blood neutrophils.30 In the present study, rheumatoid synovial fluid neutrophils separated by CFE were found to be of a consistently lower electrophoretic mobility than autologous blood neutrophils. Neutrophils within rheumatoid joints are reported to be activated as judged by their 02produc-tion28 29 and receptor expression.…”

Bimodal distribution of proteinase 3 (PR3) surface expression reflects a constitutive heterogeneity in the polymorphonuclear neutrophil pool

“…A membrane receptor for elastase, another azurophil granule enzyme closely related to PR3, has indeed be reported on PMN [28]. Furthermore, the highly basic PR3 molecule could bind to negatively charged components of the cell glycocalyx and charge heterogeneities have been described in the neutrophil pool [21]. The molecular basis of the heterogeneity among mature neutrophils is not known from this study but the stability, for one given subject, of the proportion of this PMN subset suggests that it may be under genetic control.…”

“…Various techniques of cell fractionation by density centrifugation or flow electrophoresis revealed physicochemical and functional heterogeneity in the circulating neutrophil pool [19][20][21]. Some of these variations concerned azurophil or specific granule components defining subpopulations of PMN with differences in myeloperoxidase or lactoferrin activity [22,23].…”

On the origin of low-density neutrophils

“…Besides a distribution of buoyant densities, other studies have shown functional heterogeneity within the circulating neutrophil pool of healthy humans 11,12,13 . For instance, in the studies of Eggleton et al.…”