Immunomodulatory activity of glycodelin: implications in allograft rejection

Abstract

Glycodelin is an immunomodulator, indispensable for the maintenance of pregnancy in humans. The glycoprotein induces apoptosis in activated CD4⁺ T cells, monocytes and natural killer (NK) cells, and suppresses the activity of cytotoxic T cells, macrophages and dendritic cells. This study explores the immunosuppressive property of glycodelin for its possible use in preventing graft rejection. Because glycodelin is found only in certain primates, the hypothesis was investigated in an allograft nude mouse model. It is demonstrated that treatment of alloactivated mononuclear cells with glycodelin thwarts graft rejection. Glycodelin decreases the number of activated CD4⁺ and CD8⁺ cells and down-regulates the expression of key proteins known to be involved in graft demise such as granzyme-B, eomesodermin (EOMES), interleukin (IL)-2 and proinflammatory cytokines [tumour necrosis factor (TNF)-α and IL-6], resulting in a weakened cell-mediated immune response. Immunosuppressive drugs for treating allograft rejection are associated with severe side effects. Glycodelin, a natural immunomodulator in humans, would be an ideal alternative candidate.

Keywords: IL-2; T cells; granzyme-B; immunosuppression.

Figure 1

Activity of Escherichia coli produced recombinant glycodelin (rGd). (a) Jurkat cells were treated with the indicated concentration of rGd for 24 h. Cells were then stained with annexin V‐fluorescein isothiocyanate (FITC) and propidium iodide (PI) and the apoptotic population was measured by flow cytometry (Student's t‐test P < 0·0005). (b) The alloactivated PBMCs were pretreated with 0·67 μM and 1 μM rGd for 24 h, then added to (CFSE)‐labelled human hepatocellular carcinoma expansion media (HepG2e) cells at a ratio of 10 : 1 and incubated for 4 h. After incubation, cells were harvested, stained with PI and analysed by flow cytometry (Student's t‐test, P < 0·012). Data are representative of three independent experiments.

Figure 2

Effect of recombinant glycodelin (rGd) on the in‐vivo cytotoxicity of alloactivated peripheral blood mononuclear cells (PBMCs). Nude mice were injected subcutaneously with HepG2e cells mixed with UP, AP or APG at a ratio of 1 : 10 and the tumour growth was monitored for 4 weeks. (a) Number of mice with tumour [one‐way analysis of variance (anova), P < 0·0001]. (b) The kinetics of tumour growth (one‐way anova, P < 0·048). UP = unactivated PBMCs; AP = alloactivated PBMCs; APG = alloactivated PBMCs treated with rGd. Data presented is cumulative of three independent experiments.

Figure 3

Effect of recombinant glycodelin (rGd) on the inhibition of tumour growth by alloactivated peripheral blood mononuclear cells (PBMCs). (a) Pictorial representation of the injection regimen followed for the experiment. (b) Unactivated PBMCs/alloactivated PBMCs/alloactivated PBMCs treated with two different concentrations of rGd were injected at the site of tumours and the tumour growth was measured for 15 days. UP = unactivated PBMCs; AP = alloactivated PBMCs; APG = alloactivated PBMCs treated with rGd. Data presented is cumulative of three independent experiments. [Colour figure can be viewed at http://wileyonlinelibrary.com]

Figure 4

Immune cell population in alloactivated peripheral blood mononuclear cells (PBMCs) before and after administration in mice. RNA was isolated from AP and tumours injected with AP (day 3). Real‐time polymerase chain reaction (PCR) was performed to amplify the cDNA of CD4, CD8, CD14, CD20 and CD56. The inset table tabulates the fold change in mRNA levels of each cell marker in tumour injected with AP on day 3 in comparison to AP. Data are represented as the fold expression relative to AP (before injection in mice) for each protein. AP = alloactivated PBMCs.

Figure 5

Down‐regulation of genes associated with cytotoxic activity of alloactivated peripheral blood mononuclear cells (PBMCs) by recombinant glycodelin (rGd). RNA was isolated from the tumours after injection of AP or APG. Real‐time polymerase chain reaction (PCR) was performed to detect the mRNA levels of human (a) CD4, (b) CD8, (c) interleukin (IL)‐2, (d) granzyme‐B, (e) eomesodermin (EOMES), (f) tumour necrosis factor (TNF)‐α and (g) IL‐6. The inset table tabulates the fold change in mRNA levels of each protein in APG in comparison to AP on day 3. Data are represented as the fold expression relative to the day 3 AP for each protein (Student's t‐test; *P < 0·05; **P ≤ 0·01; ***P < 0·001). AP = alloactivated PBMCs; APG = alloactivated PBMCs treated with rGd.

Figure 6

Down‐regulation of proteins associated with cytotoxic activity of alloactivated peripheral blood mononuclear cells (PBMCs) by recombinant glycodelin (rGd). Cells isolated from the tumours excised on day 15 after injection of AP or APG were stained with human antibodies specific to CD8, interleukin (IL)‐2, eomesodermin (EOMES) and granzyme‐B. Protein expression was analysed using flow cytometry. Decrease in the protein expression in APG in comparison to AP is represented as the % inhibition. AP = alloactivated PBMCs; APG = alloactivated PBMCs treated with rGd.