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M.A. Lizarralde-Iragorri et al.
tion of Lu/BCAM at the red blood cell surface using con- focal microscopy. As expected and previously shown,26 Lu/BCAM showed a punctuated expression pattern at the red blood cell surface (Figure 4A left panel; Online Supplementary Figure S4). Under oxidative conditions, there was more RBC with large fluorescent patches sug- gestive of Lu/BCAM aggregation (Figure 4A right panel; Online Supplementary Figure S4). In order to check if this was a general feature of membrane proteins under oxida- tive conditions we stained for ICAM-4, another member of the immunoglobulin superfamily, and found no differ- ence in its membrane distribution between both condi- tions (Figure 4B) indicating that oxidation targets Lu/BCAM membrane distribution through a specific mechanism.
Analysis of Lu/BCAM membrane distribution by imaging flow cytometry
We assessed Lu/BCAM membrane distribution using a high throughput approach based on imaging flow cytom- etry. RBC were classified based on the expression pattern of Lu/BCAM using the modulation feature that measures the intensity range and distribution of a fluorescent signal (see methods) (Figure 4C). Two main patterns were distin- guished in this analysis: the “Low modulation” for weak intensity dispersed spots (Spots) and “High modulation” for strong intensity big patches (Patches) (Figure 4C, last panel). Both patterns were concomitantly present in all blood samples and all conditions. We compared the pro- portion of each pattern under oxidative and control condi- tions and found less RBC with Spots and more with Patches in the presence of cumene hydroperoxide (Figure 4D; Online Supplementary Figure S5A), with no difference in Lu/BCAM global expression (Figure 4E), indicating that oxidation induces the formation of Lu/BCAM aggregates at the cell surface. As expected, based on the confocal microscopy results, oxidation did not impact the propor- tions of ICAM-4 Spots and Patches subpopulations (Figure 4F; Online Supplementary Figure S5A) or its global expres- sion at the RBC surface (Figure 4G).
Finally, we assessed Lu/BCAM distribution on SS LD and HD RBC in which both the Spots and the Patches pat- terns were found. Similar to oxidized AA RBC, the per- centage of cells with the Patches pattern was higher in HD RBC in comparison with LD RBC, while the Spots pattern prevailed in the LD subpopulation (Figure 4H; Online Supplementary Figure S5B). As expected and already report- ed, Lu/BCAM expression was lower in HD than in LD RBC (Figure 4I).
Less glycophorin C sialylation on SS red blood cells
Impact of sialic acid removal on Lu/BCAM membrane distribution
We have recently shown that Lu/BCAM can establish cis-interactions with GPC sialic acids at the RBC surface keeping it in a “locked” conformation impeding its interac- tion with laminin.32 In order to test if such interactions modulate the Lu/BCAM expression pattern, AA RBC were treated with neuraminidase (N’ase) in order to elim- inate protein sialic acids, labeled with an anti-Lu/BCAM antibody and analyzed by imaging flow cytometry. We found that loss of sialic acids resulted in less Spots and more Patches RBC (Figure 5A) with no impact on Lu/BCAM global expression level (Figure 5B), indicating that lateral interactions of Lu/BCAM with sialic acid residues impede its capacity to aggregate at the cell sur-
face. Altogether, these results suggest that oxidation may trigger Lu/BCAM release from sialic acid lateral interac- tions leading to its aggregation and activated adhesive function.
Total sialylation and specific glycophorin C sialylation levels
As HD RBC showed higher percentages of cells with the Patches pattern and that treatment of AA RBC with N’ase increases also the proportions of this sub-population, we hypothesized that increased HD RBC adhesion to laminin might result from aggregation of Lu/BCAM molecules subsequent to altered GPC sialylation. Using biotinylated lectins, we measured the sialic acid levels at the surface of RBC from nine AA and nine SS blood samples by flow cytometry and found no significant difference between the two groups (Figure 5C). We performed the same analysis on seven SCD fractionated blood samples and found less sialic acid at the surface of all HD RBC when compared to LD RBC (Figure 5D), suggesting that increased adhesion to laminin of HD RBC might result from the partial loss of interaction between Lu/BCAM and GPC at the cell surface. In order to specifically address GPC sialylation levels, we used an antibody directed against sialylated forms of GPC. Flow cytometry analysis showed less GPC sialylation levels in HD than in LD RBC (Figure 5E), while no significant difference was observed in total amounts of GPC at the cell surface as determined by flow cytometry using a sialic acid-independent anti- GPC antibody (Figure 5F). This result supported our hypothesis of increased HD RBC adhesion to laminin fol- lowing altered cis-interactions of Lu/BCAM with GPC at the cell surface.
Discussion
Oxidative stress is an important feature of SCD and plays an important role in the pathophysiology of hemol- ysis, vaso-occlusion and ensuing organ damage. In this study we investigated the relationship between oxidative stress and adhesion of RBC to laminin. We report altered protein cis-interactions at the surface of sickle dense RBC that may account for the activation of RBC adhesion in the absence of signaling events and contribute to vaso- occlusion. Using a microfluidic biomimetic chip, we con- firm the importance of the mechanical parameter in the preferential trapping of HD RBC at a single cell level, con- firming the hypothesis of the two-step model in which dense RBC contribute to the obstruction of fine blood ves- sels because of reduced deformability.13 In addition, we show that HD RBC adhere more firmly to laminin and are more resistant to shear stress than LD RBC suggesting that they would also contribute to initiate VOCs in vivo by adhering to the vessel wall even at high shear stress. This difference is probably partly due to the increased rigidity of HD RBC, as well as to the difference in cell shape between both subtypes, with a majority of very young and round reticulocytes in the LD fraction having a small- er contact surface with the capillary wall than HD RBC that are flatter cells with a larger contact surface and a smaller section facing the flow after adhesion is initiated. This is supported by the presence of long cellular tethers and of big patches of Lu/BCAM on several adhering LD RBC indicative of important membrane dynamics in this subpopulation, which is a characteristic of reticulocytes
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haematologica | 2021; 106(9)