Selective enhancement of multipotential hematopoietic progenitors in vitro and in vivo by IL-20

Abstract

In a search for novel growth factors, we discovered that human interleukin-20 (IL-20) enhanced colony formation by CD34 ؉ multipotential progenitors. IL-20 had no effect on erythroid, granulocyte-macrophage, or megakaryocyte progenitors. IL- IntroductionCytokines are important regulators of the growth and development of hematopoietic cells 1,2 ; some are currently in clinical use. In a search for novel factors that regulate hematopoiesis, colony assays were used to screen novel secreted proteins identified through bioinformatics. One protein identified was identical to . 3,4 We demonstrate that IL-20 specifically enhances the proliferation of multipotential progenitors in vitro and in vivo without effect on more lineage-restricted progenitor cells. Study design Protein productionRecombinant human , containing a C-terminal FLAG tag (Eastman Kodak, Rochester, NY) followed by 6 histidine residues (Flis), was produced in 293EBNA1 cells. 5 We captured rhIL-20-Flis on Pharmacia Chelating Sepharose FF (Amersham-Pharmacia, Piscataway, NJ). The endotoxin level was less than 5.3 EU/mg rhIL-20. Colony assays for human and murine progenitorsCD34 ϩ human bone marrow or cord blood cells were purchased from BioWhittaker (Walkersville, MD). Colony assays for granulocytemacrophage (CFU-GM), erythroid (BFU-E), and multipotential (CFU-GEMM) progenitors were as described. 6,7 Megakaryocyte (CFU-Meg) progenitor assays were carried out using MegaCult-C medium (Stem Cell Technologies, Vancouver, BC, Canada). IL-20 transgenic miceTransgenic (TG) mice were generated by established techniques. 8 Human IL-20 was overexpressed in TG mice using apolipoprotein E gene promoter. IL-20 levels in mouse serum were determined using enzyme-linked immunosorbent assay (ELISA) with antihuman rhIL-20. rhIL-20 administration to normal miceFemale BDF1 mice (8-10 weeks of age; Harlan, Indianapolis, IN) were administered rhIL-20 (5 g/mouse) subcutaneously twice a day for 10 days. At day 11, mice were killed, bone marrow and spleen cells were counted, cells were used for colony assays, and the proportion of progenitors in S-phase of the cell cycle was estimated. 7,9,10 Results and discussion IL-20 in vitroIL-20 did not stimulate colony formation, but it increased the numbers of larger-sized colonies in combination with recombinant human stem cell factor (SCF) and erythropoietin (EPO) ( Figure 1Ai-ii). Colonies cultured with IL-20 contained cells with and without hemoglobin expression. Microscopic examination of 22 individual large colonies stained with Wright-Giemsa revealed mainly erythroblasts mixed with megakaryocytes. Granulocytes and monocytes were also detected (original magnification, ϫ 400) within these colonies but were less prominent than erythroblasts and megakaryocytes . This suggested that IL-20 enhanced CFU-GEMM.Colony assays were performed with human bone marrow (BM) and cord blood (CB) CD34 ϩ cells using various cytokine combinations. IL-20 (200 ng/mL), in combination with EPO and SCF, significantly enhanced BM CFU-GEMM numbers approximate...

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