Modulation and function of progesterone induced blocking factor (PIBF) in U937 and T-PLL cell lines

Abstract

Rationale Placental CD8+ T cells exposed to progesterone produce progesterone induced blocking factor (PIBF), a 34-kDa protein that decreases natural killer cytotoxicity and prevents fetal rejection. Tumor cells could utilize PIBF for immune escape. Methods Cell lines were screened for PIBF production. U937 (monoblastic leukemia) and T-PLL (non-leukemic B-lymphocyte precursor) demonstrated high expression and were chosen for further study. Cells were cultured in RPMI 1640, 5% fetal bovine serum, 1% L-glutamine, penicillin-streptomycin until above 95% viability and count 0.5 × 106 cells/ml, and plated without and with physiologic progesterone concentrations of 5 μM and 0.5 μM, or antagonist mifepristone at 0.5 μM and 0.05 μM for 12, 24, and 48 hours. RNA was extracted (Qiagen) and semiquantitative RT-PCR conducted using primers designed with website http://www.ncbi.nih.gov and Primer 3 program for PIBF, Toll-like receptors 2, 4, 5, 7, NOD-2, IKB-α, MyD88, IRAK -1, Tollip, and LL-37. Based on RT-PCR, immunohistochemistry was performed with anti-TLR-4, TLR-2, LL-37, and PIBF antibodies and DAKO EnVision Plus staining kits. Results PIBF mRNA and protein were upregulated with progesterone and downregulated by mifepristone in both cell lines. Mifepristone decreased LL-37 protein in T-PLL. TLR4 mRNA and protein, and HNP 1-3 mRNA increased with higher amounts of progesterone in U937. No changes were observed in the other genes tested. Conclusions PIBF is expressed and modulated by progesterone in B and monocyte lineages in addition to T cells. Further investigation must differentiate whether effects on antimicrobial peptides and toll-like receptors are via progesterone receptor or through altered autocrine actions of PIBF.