Effect of Prolactin on Erythopoiesis in Diamond Blackfan Anemia: Dose-Dependent Enhancement or Inhibition of Steroid Effect in 2-Phase Erythroid Culture.

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Diamond Blackfan Anemia (DBA) is a rare congenital red cell aplasia, presenting in early infancy. The anemia is characteristically steroid responsive in the majority of cases, but eventually 40% of affected individuals are dependent on long-term transfusion and chelation programmes, underscoring the need for alternative therapies. A report by Abkowitz et al (Blood 2002; 100:2687) suggests a potential benefit of prolactin, induced indirectly by metoclopramide treatment, in a proportion of patients with steroid refractory DBA. We have therefore investigated the in vitro effect of prolactin on erythropoiesis in DBA, using a 2-phase liquid erythroid culture system in which we have previously demonstrated a severe consistent erythroid defect in DBA, and an enhancing effect of added steroids in both normal and DBA cultures. Peripheral blood mononuclear cells were cultured in serum-free medium containing 50ng/ml IL-3, 100ng/ml SCF, 1μg/ml cyclosporin A in the absence of erythropoietin (EPO) for 7 days (phase I), before transfer of non-adherent cells to phase II culture, with medium as for phase I plus 2U/ml EPO. Erythroid output was expressed as the total number of hemoglobinised cells generated after 7 days in phase II culture per cell transferred from phase I. In the absence of steroids, the addition of prolactin (PRL) 20–200ng/ml to both phases had no effect on erythroid output in normal (n=10) or DBA (n=9) cultures (table)normal (n=10)DBA (n=9)no PRLPRL 50ng/mlno PRLPRL 50ng/mlno dex5.31±1.384.87±1.390.24±0.10.18±0.07dex 10−7M10.30±1.4310.44±1.401.85±0.841.86±0.87Interaction between prolactin and dexamethasone on erythroid output in normal and DBA cultures (mean±SEM)We then studied the potential interaction between PRL and steroids, given their known synergy in lactogenesis. While PRL 50ng/ml had no overall effect on mean erythroid output in the presence of 10−7M dex (table), there was striking variation between cultures. Notably, in 3/10 normal cultures there was an apparent prolactin-induced inhibition of the normal steroid stimulatory effect. A similar phenomenon was observed in DBA cultures, with apparent synergy between 50ng/ml PRL and 10−7M dex in 3/9 (erythroid output in PRL+dex 139%, 141% and 180% of the erythroid output with dex alone), but inhibition in 3/9 (erythroid output in PRL+dex 26%, 62%, 87% of output with dex alone). Interestingly, the inhibitory effect of prolactin in DBA cultures appeared to be both more common and more pronounced at the lower PRL concentration of 20ng/ml, equivalent to the top end of the physiological range. Conversely, the stimulatory effect appeared to be more pronounced at lower concentrations of steroid (10−8M). These observations would be consistent with a dose-dependent enhancement by prolactin of steroid sensitivity, causing a left shift of the bell-shaped steroid dose response curve. The complex dose-dependence between steroids and prolactin may be of relevance to the potential therapeutic effect of metoclopramide, which increases endogenous levels of both cortisol and prolactin. Until the interactions between prolactin and steroids are more completely understood, we would advise caution in using metoclopramide in other than transfusion-dependent and steroid refractory DBA, as our results predict the risk of inhibition of steroid responsiveness in vivo, with exacerbation of anemia.