Cytotoxic activity of peripheral blood mononuclear cells in patients with endometriosis: A cross-sectional study

Abstract Background Endometriosis is believed to be associated with dysfunction of the lymphocyte population and cytotoxicity of natural killer (NK) cells, induced by the production of interleukin-2 (IL-2). Objective This study aimed to investigate T lymphocytes and NK cell activity in the peripheral blood mononuclear cells (PBMCs) of women with endometriosis. Materials and Methods PBMCs were obtained from the peripheral venous blood samples of 14 women with and without endometriosis (n = 7 for each group). Then, the PBMCs were co-cultured for 4 days and were treated with recombinant IL-2 for cytotoxic activity toward target cells (Daudi and K562 cells). The cytotoxicity activity was determined using the 51 chromium release assay before and after stimulation. Flow cytometry measurement was used to examine the expression of T lymphocytes and NK cells before and after being treated with IL-2. Results The concentration of CD3+CD28+ (co-stimulatory) was significantly lower in the endometriosis group (65.62 ± 5.38) compared to in its counterpart (50.24 ± 4.22) (p = 0.04) before stimulation. However, no significant differences were observed in any other T lymphocytes and NK cells. It was also found that there was a significant increase of CD3-CD28+ after treatment with IL-2 only in the healthy control but not in women with endometriosis. Conclusion Increased expression of CD160 and decreased CD28 play a role in inhibiting NK cell activation and T cell response in women with endometriosis.


Introduction
Endometriosis is a chronic inflammatory disorder characterized by the presence of endometrial-like tissue grows outside the uterine cavity (1). The etiopathogenesis of endometriosis is not yet well known, but one of the most prominent theories is of immunological dysfunction, causing defective immunosurveillance against aberrant autologous tissue in the peritoneal cavity, which facilitates endometriotic lesion growth in women with endometriosis (2).
The cytotoxicity of peripheral and peritoneal natural killer (NK) cells is decreased in women with endometriosis (3). However, the lower levels are due to a functional defect, not a proportion defect of the NK cells (4). One of the methods to overcome the reduced cytotoxicity is by stimulating the NK cells using stimulatory cytokine, i.e., interleukin-2 (IL-2). IL-2 stimulated NK cells which regulate the production of lymphokine-activated killer cells, indicating an increased potential against resistant tumor cells, including endometrial cells, both in-vitro and in women with endometriosis (5).
The cluster of differentiation 28 (CD28) is a costimulatory signal in T cells that modulates the function of both effector T cells and T-reg cells (6). CD28 is also a co-receptor and is involved in inducing T-cell activation. The function of CD28 on NK cells is yet unknown; however, previous studies reported that CD28 triggering in mice instigated not only NK cell proliferation but also cytotoxicity and cytokine secretion (7). Another study also demonstrated that activation of mouse IL-2-stimulated NK cells regulated the cytotoxic T-lymphocyte-associated protein 4 expression and CD28 elevation (8). These findings suggested that CD28 may be involved in the co-stimulatory pathway throughout the activation of T-cells.
Additionally, as CD28 regulates the secretion of cytokines, CD28 might be a therapeutic target to regulate the production of T-cell cytokines.
CD160 is a unique NK cell receptor that behaves as an activating receptor on CD56 dim CD16 + NK cells, which mediates natural and antibodydependent cellular cytotoxicity (9). CD160 can also promote NK cell cytotoxicity and interferonproduction in vitro and in vivo. It is also associated with CD8 T lymphocytes with cytolytic effector activity. Human CD160 expression is upregulated on CD8 T cells that lose CD28 expression (10).
However, the role of CD160-expressing T cells in women with endometriosis is poorly understood and should be investigated separately.
Given that current data related to cytotoxic activity associated with endometriosis are still limited, this study was designed to investigate T lymphocytes and their cytotoxicity markers, especially CD28 as costimulatory and CD160 as co-inhibitory, in the peripheral blood mononuclear cells (PBMCs) of women with endometriosis.

Study design
In this cross-sectional study, PBMC samples of

Subjects and sample collection
Participants were divided into 2 groups of cases and controls (n = 7/each) based on the inclusion criteria. These criteria were: 1) women of reproductive age; 2) having menstrual cycles; 3) positive for endometriosis based on histopathological and laparoscopic evidence (for the case group); 4) negative for endometriosis based on laparoscopy (for the control group); and 5) consent to participate in the study.
Women who were pregnant, using hormonal contraception, using an intrauterine device for less than 3 months, or suffering from salpingitis or malignancy were excluded. Diagnostic operative laparoscopy was performed in a total of 7 reproductive-age women whose endometriosis was classified during laparoscopy based on the revised American Fertility Society Classification (11). None of the women consumed any hormonal drug within 3 months before peripheral blood collection. Women who suffered from any endocrine disorders were excluded. Before laparoscopy, 9 mL of peripheral venous blood was collected from the women with endometriosis by venipuncture in a heparinized Hank's buffer tube and delivered to the laboratory. In addition, peripheral venous blood was also obtained from the healthy volunteers.

Isolation of PBMCs
PBMCs were obtained from the heparinized peripheral venous blood samples of the women   Data acquisition was carried out using Cell Quest software (BD Biosciences, Franklin Lake, USA).

Ethical considerations
All women gave written informed consent.

Statistical analysis
Statistical analyses were performed using the

Results
The However, no differences in any other lymphocyte subpopulations were discovered between the 2 groups before the culturing (Table II)

Discussion
The CD28, acting through phosphatidylinositol 3'kinase, is needed for T cells to increase their glycolytic response to activation (14). In this study, we found that the expression of CD3 + CD28 + was significantly lower in the peripheral blood of women with endometriosis than in the control group women. However, a higher frequency of CD160 is expressed on NK cells, CD8 + T cells, and a small subset of CD4 + T cells, and it promotes the cytotoxicity of NK cells and cytokine production (9). In this study, we found no significant difference in the expression of CD160 in the peripheral blood obtained from women with vs. without endometriosis. The expression of CD160 was elevated in the women with endometriosis compared to the control group women. In our previous study, we found that the expression of CD4 + was lower in the peripheral blood of women with endometriosis compared to their counterparts (16). The high expression of CD160 is believed to be one of the factors that plays a role in decreasing CD4 + expression in women with endometriosis because the bond between CD160 and its ligand inhibits the activation of CD4 + T cells (15).
NK cell activity is also influenced by soluble immunostimulatory cytokines such as IL-2 (17). It was found that the expression of CD28 + was higher in the women with endometriosis after being stimulated with IL-2. It has been shown that CD28 promotes T cell proliferation due to the accumulation of IL-2 (18). Thus, CD28 can deliver biochemical signals to initiate and maintain T cell responses. In this study, it was also found that there was no change in CD160 expression after being treated with IL-2 in both the endometriosis and control groups. The expression of CD160 was downregulated in the endometriosis group after stimulation. CD160 does not proliferate in response to IL-2 stimulation, which might be due to the high potential of its cytotoxic activity (19). Furthermore, a previous study using CD160 mice demonstrated that CD160 is important for interferon-production mediated by NK cells, and the sufficiency of CD160 + expression may be necessary for controlling tumor development (19).

Conclusion
In conclusion, this study, combined with a previous one (16), demonstrated that the increased expression of CD160 and decreased expression of CD28 may play a role in inhibiting NK cell activation and T cell response in endometriosis.