Effect Of Glycodelin A On Cells Of The Immune System Insights Into GdA-Induced Signaling In Monocytes, B And NK Cells
Glycodelin is a 162 amino acid dimeric, glycosylated, secretory protein of the lipocalin superfamily. Its classification as a lipocalin(carriers of small hydrophobic molecules) is based mainly on the presence of lipocalin signature motifs in its primary sequence and no ligand for this protein has been identified till date. Glycodelin has 40-55% sequence identity with β-lactoglobulin which is the second type member of the lipocalin superfamily (the first being retinol binding protein (RBP). Glycodelin is primarily a primate specific protein (though there have been isolated reports of mRNA in mice and rats) with many isoforms secreted by various tissues, predominantly of the reproductive tract. These isoforms, being the product of the same gene, are identical in primary sequence and differ only in their glycosylation due to differences in tissue origin; hence they may be better addressed as glycoforms of glycodelin. The main glycoforms of glycodelin reported till now are GdA, GdS, GdM, GdF and GdC. Each glycoform of the protein has a varied function, dictated or modulated largely by the complex glycans on its surface. GdA, the most well studied glycoform of glycodelin, is secreted by the endometrium under progesterone control and accumulates in the amniotic fluid (from where it is isolated.). GdA has been subclassifed as an immunocalin (immuno-modulatory lipocalins) due to the many immuno-modulatory functions pertaining to tissue differentiation, implantation and angiogenesis and most sifnificantly, modulation of immune responses at ehe feto-maternal interface. The fetus expresses paternal allo-antigens on its surface and would be regarded as foreign or non-self by the maternal immune system. Yet the fetus is not rejected, and is in fact protected from attack by the maternal immune system by a variety of tolerogenic mechanisms. GdA is the most abundant secretary glycoprotein of the primate uterine compartment during implantation and early pregnancy. It has been shown to have inhibitory effect on innate as well as adaptive and humoral immune responses. It inhibits the proliferation of T and B cells, Nk cytoxocity and suppresses monocyte chemotaxis. It also skews the cytokine profile from Th1 to Th2 and inhibits IL1 and IL2 secretion from mitogenically stimulated lymphocyte and mononuclear cell cultures. In our laboratory, we have demonstrated earlier that the inhibitory effect of GdA on T cell proliferation is due to apoptosis being induced. The apoptotic signaling induced by GdA was found to be caspase dependent and follows the intrinsic mitochondrial stress induced pathway of apoptosis. Having determined the effect of glycodelin A on T cells, we wanted to look at its effect on other cells playing a role in immune responses. We decided to look at its effect, if any, on the innate immune system. Chapter 1 of the thesis describes our studies on the effect of GdA on monocytes. We have looked at the effect of GdA on primary monocytes isolated from blood of healthy human volunteers and found that GdA induces apoptosis of primary monocytes and this appears to be mediated through a caspase independent pathway. The mitochondrial membrane potential of primary monocytes was lost upon GdA treatment therefore the mitochondria seem to be involved in the apoptotic cascade. As the yield of monocytes from peripheral blood is very low, further studies on the effect of GdA on monocytes were carried out using a human monocytic cell line, THP1, as a model system. We have demonstrated the GdA is able to inhibit the proliferation of these cells and also induce apoptosis in them. We also found that this signaling is partially caspase-dependent and involvement of other caspase independent pathways is possible. Further, we have shown that there is no effect of GdA on the phagocytic ability of these cells after differentiation into the macrophage lineage. However, when added before differentiation, glycodelin is able to inhibit the phagocytic ability of THP1 cells. We also found that THP1 cells were relatively resistant to GdA-induced apoptosis post differentiation into macrophages. We have also looked at the effect of GdA on B cells using primary B cells as well as a B cell line U266B1 as our model system. GdA was shown to inhibit the proliferation of primary B cells as well as of the cell line. The protein was not able to induce apoptosis in the primary cells (both activated as well as unactivated cells) as well as in the cell line. Treatment of the cells with MAP kinase inhibitors also did not render them susceptible to GdA induced apoptosis(as has been seen in the case of U937 cells). U266B1 cells remained relatively resistant to GdA-induced apoptosis even when treated for long periods. They did not undergo significant necrosis uponGdA treatment even though the proliferation of these cells was inhibited by the protein. We were surprised to find that there was loss of mitochondrial membrane potential of the cells upon GdA treatment even when there was no cell death. The reason for this is not clear. The inhibition of proliferation of B cells by GdA does not involve caspases and the signalilng induced by GdA in these cells seems to be different to that induced in T cells atleast downstream of the mitochondria as the cells cannot proliferate in presence of GdA but seem immune to further damage or apoptosis. These studies have been described in chapter 2 of the thesis. The third and final chapter of the thesis deals with our investigation into the effect of GdA on Nk cells. GdA, in an earlier report, has been shown to inhibit the activity of circulatory NK cells. However, the mechanism of this action has not been delineated. We made attempts to determine the effect of GdA on NK cells using a human NK cell line YT Indy as our model system, as isolation and culture of primary Nk cells in good numbers is difficult. Preliminary studies revealed that GdA triggered apoptosis in these cells. However, the process was found to be caspase independent. Another surprising finding was that GdA did not bring about significant loss of mitochondrial membrane potential of these cells, implying that the involvement of mitochondria in the apoptotic signaling in these cells may be at the later stages, as amplifiers rather than initiators, as has been seen in the case of T cells and monocytes.
- Biochemistry (BC)