Roles of Paracrine Factors from Bovine Luteal Cells on T Cell Function.

Abstract

Cellular interactions between T cells and luteal cells are believed to play a role in regulating homeostasis in the bovine corpus luteum (CL) during the estrous cycle. In vitro coculture of luteal cells with T cells demonstrates that luteal cells are potent stimulators of T cells. In addition to the physical contact that occurs between these cells, it is hypothesized that paracrine factors from luteal cells regulate T cell functions, particularly considering that characteristics of immune cells are influenced by cytokines. In order to test the hypothesis, luteal cells were treated with brefeldin A (BFA) to block classical secretion, which also results in upregulation of proteins secreted by unconventional pathways. In this study, conditioned media from luteal cells alone (CML) and from luteal cells treated with BFA (CMLB) were utilized to understand the paracrine interactions between T cells and luteal cells. First, both CML and CMLB significantly stimulated T cell proliferation compared to media alone with or without BFA (P < 0.01). Percentages of T cell proliferation were even higher in CMLB than those of CML, irrespective of stages of CL (midcycle and 8hPG: P < 0.01). Secondly, flow cytometry showed that protein expression of IL-2R and IL-4 in T cells cultured in CML and CMLB were significantly decreased compared to media alone, but not IFN gamma and IL-2. Thirdly, mass spectrometric analysis demonstrated that BFA treatment blocked secretion of 39 proteins, but stimulated upregulation of 9 proteins (overall fold change; −3.96 and 1.79, respectively). DeCyder analysis further identified cyclophilin A among upregulated secreted proteins in CMLB (fold increase 1.76; P = 0.03). Lastly, effects of cyclophilin A derived from CMLB on T cell proliferation was examined by addition of cyclophilin A inhibitor with different concentrations (0, 0.1 and 1 microM) during luteal cell culture. However, T cell proliferation in CMLB treated with cyclophilin A inhibitor was not changed. In conclusion, this study indicates that large numbers of proteins are secreted from bovine luteal cells by both classical and unconventional secretory pathways and luteal derived paracrine factors modulate T cell functions, but cyclophilin A is not a likely candidate. It is speculated that the cellular interactions between luteal cells and T cells may be regulated in a complex paracrine system via several secreted factors from luteal cells. This project was supported by National Research Initiative Competitive Grant no. 2008-35203-04617 from the USDA National Institute of Food and Agriculture.