Significant Biochemical, Biophysical and Metabolic Diversity in Circulating Human Cord Blood Reticulocytes

Abstract

BackgroundThe transition from enucleated reticulocytes to mature normocytes is marked by substantial remodeling of the erythrocytic cytoplasm and membrane. Despite conspicuous changes, most studies describe the maturing reticulocyte as a homogenous erythropoietic cell type. While reticulocyte staging based on fluorescent RNA stains such as thiazole orange have been useful in a clinical setting; these ‘sub-vital’ stains may confound delicate studies on reticulocyte biology and may preclude their use in heamoparasite invasion studies.Design and MethodsHere we use highly purified populations of reticulocytes isolated from cord blood, sorted by flow cytometry into four sequential subpopulations based on transferrin receptor (CD71) expression: CD71high, CD71medium, CD71low and CD71negative. Each of these subgroups was phenotyped in terms of their, morphology, membrane antigens, biomechanical properties and metabolomic profile.ResultsSuperficially CD71high and CD71medium reticulocytes share a similar gross morphology (large and multilobular) when compared to the smaller, smooth and increasingly concave reticulocytes as seen in the in the CD71low and CD71negativesamples. However, between each of the four sample sets we observe significant decreases in shear modulus, cytoadhesive capacity, erythroid receptor expression (CD44, CD55, CD147, CD235R, and CD242) and metabolite concentrations. Interestingly increasing amounts of boric acid was found in the mature reticulocytes.ConclusionsReticulocyte maturation is a dynamic and continuous process, confounding efforts to rigidly classify them. Certainly this study does not offer an alternative classification strategy; instead we used a nondestructive sampling method to examine key phenotypic changes of in reticulocytes. Our study emphasizes a need to focus greater attention on reticulocyte biology.

Plasmodium vivax: restricted tropism and rapid remodeling of CD71-positive reticulocytes

“…The distinct preference for immature reticulocytes indicated by this experiment prompted an invasion assay using more finely defined reticulocyte subsets, which were obtained as 4 populations defined by the level of CD71 expression (CD71 2 , CD71 low , CD71 medium , CD71 high ). The substantial chemical and mechanical differences between these reticulocyte subsets have been described recently by Malleret et al 5 Indeed, mere microscopic examination of subvitally stained reticulocytes from each of these 4 groups is sufficient to distinguish them, as is evident from the dark-staining reticular content and the clumping that are particularly evident in the CD71 medium and CD71 high nascent reticulocytes ( Figure 1C). However, the reinvasion assays did not reveal any statistically significant differences in the reinvasion efficacy for the 3 CD71 1 subsets, although there was a tendency for higher P vivax reinvasion with increasing CD71 levels ( Figure 1D).…”

“…Electron microscopy was conducted on reticulocytes and IRBCs using the methods outlined in Malleret et al 5 Confocal microscopy and western blot After AFM scanning, immunofluorescent assay detection of caveolin-1 was conducted on the isolates after cold acetone fixation. The primary antibodies used were caveolin-1 (#C4490, Sigma).After the addition of a second antibody (anti-rabbit immunoglobulin G fluorescein isothiocyanate [green] or Alexa 568 [red], Invitrogen) the fluorescence images were obtained using a confocal microscope (Olympus FluoView FV1000).…”

“…The red cell Duffy receptor (DARC), a known receptor for P vivax invasion, 4 is clearly not implicated because both reticulocytes and mature red cells (normocytes) express similar quantities of DARC on their surfaces. 5 On the other hand, a family of Plasmodium merozoite proteins (reticulocyte binding proteins) that preferentially bind to reticulocytes has been identified, 6 but the corresponding reticulocyte-specific receptors remain unknown. Thus, the detailed mechanism by which P vivax merozoites identify and invade human reticulocytes remains to be elucidated.…”

“…8 Notably, 1 such receptor, the transferrin receptor (CD71), is sequentially lost and becomes completely absent by the time the normocyte is formed, [9][10][11] thus making it a useful biomarker for the fine-scale age-grading of reticulocytes. 5,12,13 Most crucially, CD71 is depleted ;20 hours before the last remnants of reticular matter (commonly detected by new methylene blue [NMB] There is an Inside Blood Commentary on this article in this issue.…”

Host ICAMs play a role in cell invasion by Mycobacterium tuberculosis and Plasmodium falciparum

“…Most ICAMs are expressed on a variety of cell types such as endothelial and epithelial cells, lymphocytes, monocytes, eosinophils, and ancestral haemopoietic cells-except ICAM-4 (Landstein-Weiner blood-group antigen) that is found exclusively on RBCs. Although the level of ICAM-4 expression on erythrocyte surface decreases as they progress through various stages of reticulocyte maturation, it is expressed to at least easily detectable levels in mature erythrocytes 18,19 . Among the various ICAMs, ICAM-1 (CD54) was the first to be described and studied extensively 20 .…”