BONE-MARROW STROMA-DERIVED PROLACTIN IS INVOLVED IN BASAL AND PLATELET-ACTIVATING FACTOR-STIMULATED IN-VITRO ERYTHROPOIESIS

Cooperation between in vitro exogenous prolactin (PRL), granulocyte-ma crophage colony-stimulating factor (GM-CSF), and interleukin-3 (IL-3) at an early step of in vitro erythroid differentiation has been shown in a previous study. To gain more insight into the role of PRL in in v ivo hematopoiesis, we have now addressed the involvement of endogenous PRL in the growth of hematopoietic progenitors in a bone marrow (BM) stroma environment. The possible modulation of local PRL production by the inflammatory mediator platelet-activating factor (PAF), which is known to be produced by BM cells and to regulate pituitary PRL release , has also been evaluated. Development of burst-forming unit-erythroid (BFU-E) colonies from CD34(+) hematopoietic progenitors cultured on a BM stroma cells (BMSC) layer was slightly, but significantly, reduced in the presence of an antihuman PRL antibody. Pretreatment of BMSC wi th PAF increased the BFU-E colony efficiency of cocultured CD34(+) cel ls, and this effect was completely abrogated by the antiserum. PAF-mod ulated release of PRL by BMSC was confirmed by an enzyme-linked-immuno spot (Elispot) technique. in addition, immunoprecipitation and Western blotting experiments showed two immunoreactive products in the BMSC c ulture medium. These corresponded to the nonglycosylated (23 kD) and g lycosylated (25.5 kD) forms of pituitary PRL that are also expressed b y the B-lymphoblastoid cell line IM9-P3. Specific increase of the nong lycosylated form and decrease of the glycosylated form was observed af ter PAF treatment. Polymerase chain reaction (PCR) amplification of re verse transcribed RNA using PRL-specific primers showed the presence o f PRL message in BMSC and IM9-P3 cells. In situ hybridization experime nts with a rat PRL cDNA probe cross-reacting with human PRL mRNA confi rmed its presence in a small fraction of unstimulated BMSC and in the majority of PAF-stimulated BMSC. The enhancing effect of PAF on PRL-me diated colony formation, PRL release, and mRNA activation was countera cted by pretreating BMSC with the PAF-receptor (R) antagonist WEB 2170 . Lastly, responsiveness of BMSC to PAF was substantiated by the prese nce of the PAF-R mRNA on these cells. (C) 1997 by The American Society of Hematology.