Inhibition of erythroid progenitor cell growth by anti‐Ge3

The glycophorins C and D (GPC/D) are sialoglycoprotein components of the red cell membrane, which function to regulate shape and mechanical stability (Reid et al, 1987). Glycophorins carry the antigens of the Gerbich (Ge) blood group system including the high-incidence antigens Ge2, Ge3 and Ge4. Most infants born to women alloimmunised to Ge-antigens do not require treatment for haemolytic disease of the newborn (HDN). Generally, the infants have a positive direct-antiglobulin test, occasionally show mild jaundice, and do not require transfusion. However, two infants of one woman with anti-Ge3 had normal bilirubin values and reticulocytes at birth, but the neonatal outcomes were atypical, such that the infants developed late onset anaemia requiring transfusion a few weeks after birth (Arndt et al, 2005). Previous to these reports, similar unusual clinical features have been associated with anti-K HDN (Vaughan et al, 1994). Moreover, in vitro studies have demonstrated a suppression of erythropoiesis (Vaughan et al, 1998) and phagocytosis of early progenitor cells (Daniels et al, 2003). Arndt et al (2005) demonstrated in vitro macrophage-mediated phagocytosis of early erythroid progenitor cells by anti-Ge3. Here, we report on the inhibitory effect of anti-Ge3 on in vitro erythroid progenitor cell growth. Two examples of maternal anti-Ge3 associated with HDN were evaluated; one with a titre of 256 (Arndt et al, 2005) and the other with a titre of 32, together with a murine monoclonal anti-GPC. Cord blood mononuclear cells (1·5 × 105) were cultured in MethoCult media (Stem Cell Technologies, Vancouver, BC, Canada) supplemented with 3 U/ml of erythropoietin. Cultures were established in the presence of 20% (v/v) maternal sera, Iscove's modified Dulbecco's medium (IMDM) containing 10 μg/ml of murine immunoglobulin (Ig) G1 monoclonal anti-GPC (Abcam Ltd, Cambridge, UK), or an irrelevant murine IgG1 in IMDM as a control (Serotec, Raleigh, NC, USA). Polyclonal anti-K (Dominion Biological Limited, Dartmouth, NS, Canada) in IMDM and maternal anti-D serum (titre 256) served as positive and negative controls respectively. MethoCult media containing erythropoietin was used to determine the maximum colony growth. Sera were evaluated in triplicate, and the number of erythroid colony-forming units (CFU-E) and burst-forming units (BFU-E) were evaluated after 7 and 14 d of incubation at 37°C/5% CO2 respectively. The statistical significance for the mean number of colonies per well compared with culture media without antibodies was evaluated using a two-sided Student's t-test, with P < 0·05 considered to be significant. Both maternal sera containing anti-Ge3, the murine anti-glycophorin C and anti-K significantly inhibited BFU-E and CFU-E growth (Fig 1). The maternal anti-D and the murine isotype (negative controls) had no effect. The inhibitory effect was greater for the high titre anti-Ge3. Data showed that the inhibitory effect of blood group antibodies on erythroid progenitor cell growth, seen with anti-K, was also observed for antibodies that target GPC/D. It is important to note that both the Kell glycoprotein and the glycophorins are expressed on early erythroid progenitor cells (Bony et al, 1999). Our observations provide additional evidence that the postpartum course of infants with maternal anti-Ge3 should be monitored closely for a few weeks regardless of the initial haemoglobin and especially when they have a low reticulocyte count and no hyperbilirubinemia. Equally important, pregnancies complicated by anti-Ge should be managed like anti-K, i.e. the antibodies should be considered to be potentially clinically significant regardless of the titre. The effect of anti-glycophorins on erythroid progenitor cell growth. Maternal anti-Ge3 [titre = 256 (T256) or titre = 32 (T32)], a murine monoclonal anti-glycophorin C, and anti-K (positive control) inhibited CFU-E evaluated on day 7 and BFU-E on day 14, relative to culture media without antibodies (Ge3 + K + D + cord blood). Sera containing anti-D and a murine isotype control had no effect. Results are expressed as the mean (± SD) number of colonies as a percentage of the maximum. In summary, phagocytosis of early erythroid cells expressing GPC/D and the concomitant inhibition of erythroid colony formation are associated with late onset neonatal anaemia. However, the relative contributions of early erythroid progenitor cell phagocytosis and growth inhibition to disease outcome remain unknown.