rIL-3

Interleukin-33 (IL-33) promotes migration of cancer cells through downregulating the expression of E-cadherin. Previous studies have demonstrated that IL‑33 stimulates the proliferation of trophoblasts. However, the effect of IL‑33 on the adhesion and invasion of trophoblasts has not been investigated in detail. In the present study, the expression of IL‑33 and its receptor, IL‑1 receptor‑like 1 (ST2), was examined in villi from women during early pregnancy using immunohistochemistry. ST2 expression on human trophoblast and choriocarcinoma cell lines JAR, BeWo, JEG3 and HTR8 was confirmed by flow cytometry (FCM) assay. The effect of recombinant human IL‑33 (rhIL‑33) on adhesion, invasion and associated molecules was analyzed by cell adhesion, Matrigel invasion and FCM assays. The current study identified that human trophoblasts expressed IL‑33 and ST2. RhIL‑33 inhibited trophoblast invasion and adhesion, and decreased adhesion and invasion‑associated molecules such as integrin α4β1 and CD62L. Therefore, these results suggest that IL‑33 may serve an important role in limiting invasion and implantation of trophoblasts by adhesion and invasion‑associated molecules, contributing to the formation of the placenta and maintenance of normal pregnancy during early pregnancy.

Recombinant human interleukin-3 (rhIL-3) is a multiple hematopoietic growth factor, which enhances stem cell expansion and hematopoiesis regeneration in vitro and in vivo, when administrated in combination with other cytokines. However, the structure-function study of rhIL-3 remains rarely studied, so far. The purpose of this study was to recognize the short peptide with similar function as rhIL-3, and assess the hematopoietic efficacy in umbilical cord blood (UCB) stem cell culture as well.

Two novel monoclonal antibodies (mAb) (C1 and E1) were generated against rhIL-3 using hybridoma technique. Eleven short peptides were depicted and synthesized to overlap covering the full length sequence of rhIL-3. ELISA was employed to distinguish the antibody-binding peptide from the negative peptides. In addition, the multi-potential hematopoiesis capabilities of the positive peptides were evaluated by adding 25 ng/mL of each peptide to the culture medium of hematopoietic stem cells (HSCs) derived from UCB. Total nucleated cell number and the CD34(+) cell number from each individual treatment group were calculated on day 7. Correlated antibodies at 0.5 or 2 molar fold to each peptide were also tested in the stem cell expansion experiment, to further confirm the bioactivity of the peptides.

Two peptides were recognized by the novel generated antibodies, using ELISA. Peptide 3 and 8 exhibited comparable hematopoiesis potentials, with 25.01±0.14 fold, and 19.89±0.12 fold increase of total nucleated cell number on day 7, respectively, compared with the basal medium control (4.93±0.55 fold). These biological effects were neutralized by adding the corresponding mAb at a dose dependent manner.

Our results identified two specific regions of rhIL-3 responsible for HSC proliferation and differentiation, which were located from 28 to 49 amino acids (P3), and 107 to 127 amino acids (P8), respectively. The short peptide 3 and 8 might act synergistically, which could serve as an economic substitute to rhIL-3 in research laboratory.