Expression of cathepsin proteinases by mouse trophoblast in vivo and in vitro

Abstract

Implantation and placentation in the mouse requires successful invasion of the uterine wall by primary and secondary trophoblast giant cells. Their invasive nature depends in part on the upregulation of proteinases for the phagocytosis and extracelluar digestion of maternal cells and matrix materials. The work reported here studies the expression of cathepsin proteinases during secondary trophoblast differentiation, and compares the expression patterns to fully differentiated day 8.5 primary trophoblast giant cells. Cathepsins B (CB), L (CL), and D (CD) were found to be upregulated during trophoblast differentiation in vivo at the message and protein level producing expression patterns equivalent to those of primary trophoblast. Invasive trophoblast cells expressed higher levels of the processed or active forms of the enzymes, coinciding with the period of trophoblast phagocytosis of maternal blood, decidual cells, and matrix materials. Trophoblast differentiation in vitro showed a similar upregulation of cathepsin enzymes. The enzymes were localized to heterogeneous vesicles that resembled both lysosomes and heterophagic vesicles. The presence of a large lysosomal population within the giant cells was confirmed by vital staining with acridine orange. Analysis of trophoblast‐conditioned media also demonstrated secreted forms of CB and CL. The results suggest that cathepsin enzymes may contribute to trophoblast invasion not only through the intracellular breakdown of molecules phagocytosed by trophoblast cells, but also by the extracellular digestion of matrix molecules and activation of other pro‐enzymes. Dev Dyn 1999;216:374–384. ©1999 Wiley‐Liss, Inc.

Serpin-6 Expression Protects Embryonic Stem Cells from Lysis by Antigen-Specific CTL

“…LCMV-specific cytotoxicity of primary CD8 ϩ CTL was assessed in a 4-h standard chromium release assay. Target cells were infected 48 h before the assay with LCMV at a multiplicity of infection of 0.01 or loaded for 1 h with the synthetic peptide representing the immunodominant H-2D b -restricted epitope (gp [33][34][35][36][37][38][39][40][41] ) of the LCMV glycoprotein 1 at the indicated concentrations.…”

“…Ten micrograms of each sample was separated on SDS-polyacrylamide gel and immunoblotting was performed as described previously (23) Ϫ6 M for 1 h before non-bound peptide was thoroughly washed away. B, Target cells were pretreated for 48 h with 20 ng/ml IFN-␥ before loading with epitope gp [33][34][35][36][37][38][39][40][41] . These cytotoxicity assays were repeated three (A) or five (B) times with similar results.…”

“…To bypass possible defects in Ag processing and presentation in ES and EB cells, MHC class I molecules were externally loaded with the gp [33][34][35][36][37][38][39][40][41] epitope. Preincubation of syngeneic C57BL/6-SV fibroblasts with the synthetic peptide resulted in efficient killing by LCMV-specific CD8 ϩ CTL, while undifferentiated ES cells loaded with gp [33][34][35][36][37][38][39][40][41] were lysed just slightly above background levels, and differentiated cells of EB loaded with gp [33][34][35][36][37][38][39][40][41] were not significantly lysed by the same effector cells (Fig. 4A).…”

“…To test whether lack of killing of ES and EB-derived cells by CTL was secondary to low-level MHC I expression, ES and EBderived cells were pretreated with IFN-␥ for 48 h and subsequently loaded with the LCMV gp [33][34][35][36][37][38][39][40][41] peptide. However, IFN-␥-induced up-regulation of MHC I resulted only in day 8 EB-derived cells in clearly increased lysis (Fig.…”

“…ϩ target cells required an ϳ100-fold load of gp [33][34][35][36][37][38][39][40][41] to induce secretion of comparable amounts of IFN-␥ (Fig. 6D).…”