Differential interaction between DARC and SDF-1 on erythrocytes and their precursors

The Duffy Antigen Receptor for Chemokines (DARC) is expressed on erythrocytes and on endothelium of postcapillary venules and splenic sinusoids. Absence of DARC on erythrocytes, but not on endothelium, is referred to as the Duffy negative phenotype and is associated with neutropenia. Here we provide evidence that stromal cell-derived factor 1 (SDF-1), the chemokine that restricts neutrophil precursors to the bone marrow, binds to erythrocyte progenitors in a DARC-dependent manner. Furthermore, we show that SDF-1 binding to DARC is dependent on the conformation of DARC, which gradually changes during erythroid development, resulting in the absence of SDF-1 binding to mature erythrocytes. However, SDF-1 binding to erythrocytes was found to be inducible by pre-treating erythrocytes with IL-8 or with antibodies recognizing specific epitopes on DARC. Taken together, these novel findings identify DARC on erythrocyte precursors as a receptor for SDF-1, which may be of interest in beginning to understand the development of neutropenia in situations where DARC expression is limited.

binding to erythrocytes and its precursors we used biotinylated SDF-1 antibody listed in supplementary table 1 followed by streptavidin-647 conjugation. Afterwards, we took along nuclear staining (Hoechst), anti-transferrin receptor (anti-CD71-FITC) and glycophorin-A staining (anti-CD235a-PE) to distinguish between the various stages of erythroid development. In case we had to perform additional stainings, such as in the case of determining Fy epitope exposure (e.g. Fy a , Fy b , Fy 3 or Fy 6 ) on SDF-interacting and non-interacting reticulocytes, we switched the order to ensure antigen-specific staining. In short, we first stained for Fy epitopes followed by either secondary anti-human-405 (for Fy a and Fy b ) or anti-mouse-405 (for Fy 3 or Fy 6 ) after which we stained for SDF followed by streptavidin-647 conjugation, after which we again took along anti-CD71-FITC and anti-235a-PE).
To further ensure antigen-specific staining we took along the appropriate IgG isotype controls. Exogenous addition of SDF-1 to erythroid cells was performed at 37 °C for 30 minutes whereas staining was performed at 4 °C. Flow-cytometry analysis was performed on LSRII + HTS and data were analysed by FACSDiva software (BD Biosciences, Franklin Lakes, USA).

Results and Discussion
Reticulocytes bind SDF-1. To test if DARC differentially binds SDF-1 during erythrocyte maturation we first assessed membrane-bound SDF-1 on erythrocytes and reticulocytes. We found that the most immature CD71 high reticulocytes in the circulation, and to a lesser extent CD71 low reticulocytes, but not erythrocytes, bind SDF-1 (Fig. 1a). These data were further supported by imagestream analysis (Fig. 1b). We found that SDF-1 binding to bone marrow reticulocytes (the most immature reticulocytes) was even higher in comparison to reticulocytes and erythrocytes from peripheral blood (Fig. 1c). In contrast, various other chemokines that have previously been described, similar to SDF-1, not to interact with erythrocytes 5 , did not bind reticulocytes (Fig. 1d). In agreement with the flow cytometry data, western blot analysis showed that CCL3, CCL4 and CCL21, in contrast to SDF-1, were not detected on the membranes of both erythrocytes and reticulocytes (Fig. 1e). Note that recombinant SDF-1 runs at a lower molecular weight as compared to endogenous SDF-1 (See Supplemental Table 1 for specifics). Importantly, we found that SDF-1 did not bind to reticulocytes isolated from Fy −/− individuals ( Fig. 1f), suggesting that SDF-1 binding to reticulocytes depends on expression of DARC. In addition and in support of this hypothesis, CCL2, a known DARC-binding chemokine 17 , inhibits SDF-1 binding to DARC + reticulocytes ( Fig. 1g). Thus we suggest that SDF-1 binds to DARC on immature CD71 + reticulocytes and this interaction is lost upon reticulocyte maturation to erythrocytes.
Erythrocyte precursors bind SDF-1. DARC is expressed in erythroid precursors 18 . As reticulocytes bind SDF-1 in a DARC-dependent manner this may suggest that erythrocyte precursors may also bind SDF-1 through DARC. In vitro cultured erythroid precursors can be ranked from early pro-erythroblasts to late enucleated reticulocytes. We defined several erythroblast populations based on CD71 and CD235a expression 19 ( Fig. 2a,b). The bona fide receptor of SDF-1 is CXCR4, which expression was quickly downregulated at the onset of erythroblast differentiation (Fig. 2a,c). DARC was already expressed on erythroblasts and expression was maintained during differentiation (Fig. 2c). Next we assessed SDF-1 binding dynamics during erythroblast differentiation. Strikingly, we found that almost all erythrocyte precursors were capable of binding SDF-1. This was found to gradually decrease during their maturation into reticulocytes (Fig. 2d, Suppl. Fig. 1a). These results suggest that SDF-1 binding to erythrocyte precursors is dependent on the erythroid maturation stage and that CXCR4 is not involved in this as CXCR4 expression is quickly downregulated at the onset of differentiation.
DARC epitope exposure is influenced upon SDF-1 binding. The region between the N-terminal domain that carries the Fy 6 epitope and the fourth extracellular domain within DARC, is required to switch to an active chemokine-binding pocket 20 (Fig. 3a). In addition, we previously reported increased accessibility of the DARC Fy6 epitope within immature reticulocytes compared to erythrocytes 12 . Therefore we assessed if the accessibility of specific epitopes within DARC, and in particular epitope Fy6, is increased on SDF-1-interacting reticulocytes from the circulation. We found an increased association of Fy 6 epitope recognizing antibodies on SDF-interacting reticulocytes, as compared to those that did not contain membrane bound SDF-1 (Fig. 3b). To a significantly lesser extent this was also observed for Fy a . SDF-1 binding did not affect the association of antibodies to Fy b , Fy 3 or the control CD235a. This suggests that increased exposure of the Fy 6 epitope within DARC detected on both erythrocytes as well as on reticulocytes. SDF-1 (~10.6 kDa) however was detected only on reticulocytes and not on erythrocytes. The molecular weight difference between recombinant and reticulocyte endogenous SDF-1 may result from post-translation modification or dimerization of SDF-1. (f) Quantification of SDF-1 binding to erythrocytes and reticulocytes from DARC negative and positive individuals (n = 3-12, unpaired T-test; *P < 0.05;**P < 0.01). (g) Effect of exogenous addition of SDF-1 to erythrocytes and reticulocytes in the absence and presence of equimolar levels of CCL2 (n = 5, paired T-test; *P < 0.05

SDF-1 binding to DARC on erythrocytes and reticulocytes is inducible. Next we investigated if
an antibody specific to the Fy6-epitope would interfere with SDF-1 binding. Indeed, blocking the Fy 6 epitope prior to exogenous addition of SDF-1 resulted in a significant reduction of SDF-1 binding (Fig. 4a). This finding suggests that exposure of the Fy6 epitope is altered on immature reticulocytes and may be required for SDF-1 binding. Unexpectedly, in contrast to decreased SDF-1 binding to reticulocytes due to blocking with anti-Fy 6 antibody, both Fy a and Fy b antibody binding led to increased SDF-1 binding. In addition, pre-treatment with IL-8, a chemokine known to bind to DARC, also increased SDF-1 binding to reticulocytes (Fig. 4b). This finding suggests www.nature.com/scientificreports www.nature.com/scientificreports/ that the binding of an antibody or a chemokine can cause changes in the exposure of specific parts within DARC on reticulocytes, allowing the binding of SDF-1. This led us to hypothesize that these antibodies/chemokines may render erythrocytes that normally are not capable of binding SDF-1 permissive to binding SDF-1. Indeed, binding of Fy a and Fy b antibodies as well as IL-8 to DARC allowed SDF-1 to bind to erythrocytes (Fig. 4c). These findings underscore that the ability of DARC to bind SDF-1 is not indefinitely lost on erythrocytes and can be modulated by chemokines. Together, these results show that DARC affinity for SDF-1 changes during reticulocyte maturation into erythrocytes and strongly suggests that SDF-1 binding is dependent on DARC conformation 8,21 . Next to performing these competition experiments on erythrocytes and reticulocytes, we also assessed the ability of anti-Fy 3 , anti-Fy 6 and IL-8 to interfere in the interaction between DARC and SDF-1 in erythroid precursors. We found that the percentage of erythroid precursors that bound SDF-1 upon co-incubation with anti-Fy 3 , anti-Fy 6 and IL-8 was not affected (Fig. 4d). However, as observed in reticulocytes, a significant relative reduction of SDF-1 bound per erythroid precursor was observed, suggesting competition (Fig. 4e). In contrast to reticulocytes from circulation, anti-Fy a and anti-Fy b antibodies had a strong negative effect on SDF-1 binding to cultured erythroid precursors. This inhibiting effect became less apparent as erythroid precursors matured from CFU-e to orthochromatic/polychromatic erythroblasts, which is reflected both in the percentage of cells interacting with SDF1 (Fig. 4d) as well as the amount of SDF-1 bound (Fig. 4e), resembling more the circulating reticulocytes (Fig. 4b). These results underscore that SDF-1 binding to DARC, and the ability to induce or prevent this interaction from occurring, is dependent on the stage of maturation.
Taken together, we report the novel finding that, during maturation, erythroid precursors gradually down regulate the ability to bind SDF-1, and we propose that this binding is DARC-dependent. The antibody and chemokine binding assays indicate that, during terminal differentiation into the erythrocyte stage, the decreased binding of SDF-1 may be due to a conformational switch in DARC, which is a phenomenon that has been shown to account for the selective invasion of reticulocytes by Plasmodium vivax 12 . Recent research in a murine knockout model showed that DARC expression on erythrocyte progenitors is essential for the retention of immature neutrophils in the bone marrow and the spleen. Here we speculate that this retention could be dependent on SDF-1, the chemokine that is mostly known for restricting neutrophil precursors to the bone marrow. SDF-1 www.nature.com/scientificreports www.nature.com/scientificreports/ association to erythrocyte progenitors in a DARC-dependent manner may change the availability of SDF-1 within the bone marrow possibly affecting hematopoietic progenitor and effector cell (e.g. neutrophils) migration. This may begin to explain the neutropenia in Duffy negative individuals. It would be of great interest to further study the function of DARC in neutrophil localization and mobilization in and from the bone marrow.