Glycodelin A triggers mitochondrial stress and apoptosis in T cells by a mechanism distinct and independent of TCR signaling
Introduction
The fetus expresses paternal alloantigens and would be regarded as non-self or foreign by the maternal immune system, but for a set of synergistic mechanisms that protect it from rejection and thereby sustain pregnancy. Among these, immunomodulation at the feto-maternal interface is achieved by altered cytokine profiles and suppression of immune cells capable of attacking the fetus. The decidua contains a diverse population of immune cells including lymphocytes, uterine NK cells and monocytes. Induction of apoptosis in these cells by Fas ligand (FasL) and tumour necrosis factor (TNF) family ligands expressed by the trophoblast plays a key role in fetal tolerance (Veenstra van Nieuwenhoven et al., 2003). In addition, soluble mediators such as indoleamine 2,3-dioxygenase also facilitate lymphocyte apoptosis at the decidua (Terness et al., 2007). Apoptosis of lymphocytes in the uterine compartment is therefore a critical mechanism towards immuno-tolerance of the fetus. Indeed, disturbance in programmed cell death of activated T cells in the human decidua has been implicated in pregnancy loss (Jerzak et al., 1998).
Glycodelin A (GdA), also known as Placental protein 14 (PP14), is a pregnancy-associated immunomodulatory factor of the primate uterine compartment. It is the most abundant progesterone-regulated secretory glycoprotein of the uterus at the time of implantation and early pregnancy (Seppala et al., 2002). GdA is inhibitory to T cell (Bolton et al., 1987, Rachmilewitz et al., 1999, Mukhopadhyay et al., 2001) and B cell (Yaniv et al., 2003) proliferation, NK cell activity (Okamoto et al., 1991) and induces apoptosis in activated T cells (Mukhopadhyay et al., 2001). It is also reported to stimulate the Th2-type cytokine profile (Mishan-Eisenberg et al., 2004), and inhibit interleukins IL-2 (Bolton et al., 1987) and IL-1 (Pockley and Bolton, 1990) production from mitogenically stimulated lymphocytes and mononuclear cell cultures. Furthermore, it has been proposed to desensitize T cell receptor (TCR) signaling (Rachmilewitz et al., 2001), by its association with the tyrosine phosphatase CD45 (Rachmilewitz et al., 2003). Due to its pleiotropic nature, namely its diverse activities on different immune cell types, and the spatio-temporal restriction of its expression by progesterone, GdA is being increasingly recognized as a mechanism towards fetal tolerance. In agreement, subnormal levels of GdA are correlated with implantation failure and infertility (Mackenna et al., 1993), habitual abortion (Tulppala et al., 1995) and recurrent miscarriage (Dalton et al., 1998).
Our laboratory has focused on the T cell inhibitory activity of GdA. We have shown that GdA induces apoptosis in activated T cells (Mukhopadhyay et al., 2001) and that the protein backbone is necessary and sufficient for this activity (Jayachandran et al., 2004). We have also shown that the N-linked glycosylation on GdA modulates accessibility of the active site on the protein (Mukhopadhyay et al., 2004, Jayachandran et al., 2006, Poornima and Karande, 2007). From the evidence presented earlier, it can be surmised that the apoptogenic activity of GdA contributes to its role in fetal tolerance. Therefore, the mechanism by which GdA induces apoptosis in T cells is of interest and is the subject of the present study. We report here that GdA-induced apoptosis does not involve death receptor signaling. Rather, GdA triggers mitochondrial stress and the intrinsic pathway of programmed cell death. Further, we observe that stress-induced apoptosis initiated by GdA is not a consequence of desensitization of TCR signaling, and does not require CD45. In fact, GdA-induced apoptosis proceeds by a mechanism that is distinct from and independent of the T cell activation pathway, and novel from those so far reported at the feto-maternal interface.
Section snippets
Reagents
HISTOPAQUE, phytohemagglutinin (PHA), phorbol myristate acetate (PMA), ionomycin, JC-1, valinomycin, DCFH-DA, BAPTA-AM, fumonisin B1, sphingosine-1-phosphate and recombinant human IL-2 were obtained from Sigma–Aldrich. Fas ligand (FasL), anti-Fas monoclonal antibody CBL-537, Z-VAD-FMK, Z-LEHD-FMK, SP600125, SB203580, PD98059 and PP2 were purchased from Calbiochem. Z-VDVAD-FMK was obtained from Enzymes Systems Products. Phycoerythrin conjugated anti-CD69 mAb was obtained from BD Pharmingen.
Cells and cell lines
The
GdA induces mitochondrial stress and activates the intrinsic pathway of apoptosis
To delineate the molecular events initiated by GdA, we first examined the requirement of caspases for the apoptotic program. Jurkat cells were treated with GdA in the presence or absence of the pan caspase inhibitor Z-VAD-FMK (Fig. 1). Inhibitor treated cells were completely resistant to apoptosis (Fig. 1C) confirming that GdA-induced apoptosis is caspase dependant. We have previously reported the involvement of caspase 3 in the pathway (Mukhopadhyay et al., 2001).
Apoptosis can be initiated by
Discussion
Immune responses play a pivotal role during pregnancy as the maternal immune system must accept the semi-allogeneic fetus. This was first recognized by Billingham and Medawar (1953) when the concept of the fetal allograft was presented to explain the immunological relationship between the mother and the fetus. Several integrated mechanisms of various origins and modes of action have since been identified to play key roles in establishing maternal immune tolerance towards the fetus (Veenstra van
Acknowledgements
This work was supported by grants from the Department of Biotechnology, Government of India. We thank Dr. Omana Joy for assistance with the FACScan analysis.
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Recipient of a senior research fellowship from the Council of Scientific and Industrial Research, Government of India.