Changes in the properties of normal human red blood cells during in vivo aging

Abstract

The changes in red blood cells (RBC) as they age and the mechanisms for their eventual removal have been of interest for many years. Proposed age-related changes include dehydration with increased density and decreased size, increased membrane IgG, loss of membrane phospholipid asymmetry, and decreased activity of KCl cotransport. The biotin RBC label allows unambiguous identification of older cells and exploration of their properties as they age. Autologous normal human RBC were labeled ex vivo and, after reinfusion, compared with unlabeled RBC throughout their lifespan. RBC density increased with age, with most of the change in the first weeks. Near the end of their lifespan, RBC had increased surface IgG. However, there was no evidence for elevated external phosphatidylserine (PS) even though older RBC had significantly lower activity of aminophospholipid translocase (APLT). KCl cotransport activity persisted well past the reticulocyte stage, but eventually decreased as the RBC became older. These studies place limitations on the use of density fractionation for the study of older human RBC, and do not support loss of phospholipid asymmetry as a mechanism for human RBC senescence. However, increased levels of IgG were associated with older RBC, and may contribute to their removal from the circulation.

Figure 1

Confocal analysis of fixed and permeabilized RBC (subject NDM2) 78 days after reinfusion. A cell entirely coated with biotin (green) and a different cell with a small biotin cluster are shown in the first picture. A different cell in the field is seen to contain 4 clusters of IgG (second picture). The same cell that has IgG has a strong band 3 label (third picture). The fourth picture shows the overlay of all labels.

Figure 2

IgG on old RBC. A: (NDM1, day 126) At day 126 the biotinylated cells appear misshapen, and are all IgG labeled. B: IgG coated >100 day old biotinylated RBC isolated using magnetic beads derivatized with streptavidin (left panel), and the nonisolated aliquot from the same sample (right panel).

Figure 3

Age-dependent cellular density changes for subjects NDM1 and NDM2. RBC were separated by density using a discontinuous gradient. The top charts in each column show the average percent cells (± 1 sd) in each fraction for the unlabeled RBC at all time points (N = 16 for NDM1, N = 13 for NDM2). The bottom four charts in each column show the density distributions for labeled RBC at selected time points. Day 0 is immediately after reinfusion.

Figure 4

The mean forward scatter FS for unlabeled (◇) and labeled (■) RBC as a function of time since reinfusion.

Figure 5

Age-dependent cellular external PS. The x-axis shows the percentage of the labeled RBC that have been removed from the circulation. The y-axis shows the percentage of labeled RBC with external PS normalized by subtracting the concurrent percentage of unlabeled RBC with external PS. Data on 4 subjects (open symbols) are compared to theoretical curves (lines) calculated assuming RBC with external PS remain in the circulation for 1, 0.5, or 0.1 days prior to removal.

Figure 6

Age-dependent cellular change in APLT activity. The fraction of NBD-PS transferred from the outer to the inner leaflet in biotin-labeled RBC was determined and expressed relative to the fraction in unlabeled RBC in the same sample. The effects of compounds that inhibit APLT activity (2 µM NEM and 10 mM vanadate) are also shown as controls.

Figure 7

A: KCC mediated volume reduction in mature RBC. Advia 120 histograms of CHC of nonreticulocyte AA RBC are shown for a single experiment representative of six others. Cells were swollen with nystatin to CHCM = 24 g/dl (t = 0) then incubated at 37°C without (control) or with 600 mM urea (to activate KCC) for the times indicated. Cells were washed in cold HBS and kept on ice until Advia analysis. Vertical markers in each histogram represent CHC of 27 and 41 g/dl. B: Blood samples were taken 7 days or 62 days after reinfusion of BRBC, treated with nystatin, and incubated with urea to activate KCC as above. Density distributions pre- and posturea incubation are shown for unlabeled RBC (gray bars) and labeled BRBC (black bars). At 7 days, when the BRBC are only slightly older than the RBC, a similar shift in density is apparent in both RBC and BRBC. After 62 days a density shift upon KCC activation is again apparent in the steady state RBC, but does not occur in the BRBC, which are at least 62 days old. Lower panes (62 days only) indicate that removal of Cl eliminates the density shift as would be expected for KCC. Data represents a single experiment, replicated independently in another subject. Fx1 = 1.080 g/cc; Fx2 = 1.085; Fx3 = 1.090; Fx4 = 1.095; Fx5 = 1.100 (C) Since hydration changes are subject to experimental variability as discussed in the text, the changes in BRBC were normalized to the changes in steady state RBC at each timepoint by calculating the ratio of the percentage BRBC exhibiting urea-stimulated, KCC-mediated density shift to that of RBC (see text for details). Data from two subjects indicate that for approximately the first half of the red cell lifespan KCC can be activated under these conditions. Data from two subjects are shown.