Effect of endometriosis on white blood cell subpopulations in peripheral blood and peritoneal fluid of baboons.

In women with endometriosis, changes in peripheral blood and peritoneal fluid white blood cell (WBC) populations have been reported, but it is known whether these alterations are casually related to or a consequence of endometriosis. The purpose of this study was to test the hypothesis that peripheral blood and peritoneal fluid WBC populations are altered in baboons with spontaneous and induced endometriosis compared to animals without disease. Peripheral blood and peritoneal fluid samples were obtained at laparoscopy from 60 baboons with a normal pelvis (n = 23), spontaneous endometriosis (n = 19) and induced disease (n = 12), luteal phase (n = 20), pregnancy or nursing (n = 11) and in non-cycling animals (n = 8). The WBC concentration was analysed with a Coulter counter and fluorescent antibody cell separation (FACS) analysis was used to measure cluster designation (CD)2, CD4, CD8, interleukin (IL)2R and leucine (Leu) M5 subsets. In peripheral blood, the percentage of CD4+ and IL2R+ cells was increased in baboons with stage II-IV spontaneous or induced endometriosis, suggesting that alterations in peripheral blood WBC populations may be an effect of endometriosis. In peritoneal fluid the WBC concentration and percentages of Leu M5+ macrophages and CD8+ lymphocytes were only increased in baboons with spontaneous endometriosis and not in animals with induced disease, suggesting that alterations in peritoneal fluid WBC populations may lead to the development of endometriosis. In summary, the results of this study suggest that peripheral blood and peritoneal fluid immune cell populations are affected in baboons with endometriosis.

CO2-laser excision of endometriosis does not improve the decreased natural killer activity.

OBJECTIVE: We recently described a decreased lymphocyte-mediated cytotoxicity to autologous endometrium and to natural killer-sensitive targets in women with endometriosis. The purpose of the present study was to investigate whether the defect remained after CO2-laser excision of the endometriotic lesions. DESIGN: In 15 women with endometriosis the cytotoxicity assays were performed before and three to four months after complete removal of the endometriotic lesions with CO2-laser excision. CA-125 serum levels were measured before and after laser laparoscopy, in order to confirm that endometriosis had been treated properly. RESULTS: After removal of the endometriotic lesions the decreased natural killer activity (K562-assay) and the impaired cytotoxicity of autologous and even heterologous lymphocytes to the endometrium remained unchanged, and those cytotoxicity assays were still significantly decreased compared to the women without endometriosis. CA-125 serum concentrations, however, were still significantly decreased three to four months after CO2-laser excision of the endometriosis. (paired Student's t-test p < 0.007). CONCLUSIONS: These data suggest a primary deficiency in natural killer activity in women with endometriosis and are in accordance with our clinical experience as endometriosis is relapsing frequently after treatment.

Transforming growth factor-beta activity is increased in peritoneal fluid from women with endometriosis.

OBJECTIVE: To investigate the presence of transforming growth factor-beta in peritoneal fluid of women with and without endometriosis. METHODS: Fifty-two peritoneal fluid samples, obtained during laparoscopies performed for tubal ligation (n = 10), infertility (n = 38), or pain (n = 4), were examined for the presence of transforming growth factor-beta using the Mv1Lu cell growth inhibition assay. At laparoscopy, 26 women had endometriosis. The other 26 women had no endometriosis; 16 of them had infertility, and ten who had no pelvic pathology at tubal sterilization served as fertile controls. RESULTS: The concentration of transforming growth factor-beta was increased in the peritoneal fluid from women with endometriosis (11.4 +/- 3.3 ng/mL) compared to both the fertile control group without endometriosis (1.1 +/- 0.29 ng/mL) and the infertile control group without endometriosis (3.6 +/- 1.4 ng/mL). Twenty-five of the 52 women (48%) demonstrated levels of transforming growth factor-beta higher than 2 ng/mL. Patients with endometriosis were significantly more likely to have elevated concentrations of transforming growth factor-beta than were women without endometriosis (16 of 26, 61.5%, versus nine of 26, 34.6%). CONCLUSIONS: These findings demonstrate the presence of transforming growth factor-beta in peritoneal fluid. Elevated levels in women with endometriosis could be important in the pathophysiology of this disease.

Lymphokine-activated killer activity in women with endometriosis.

The aim of the present study was to investigate whether women with endometriosis displayed a decreased lymphokine-activated killer (LAK) activity. In 15 women with and 7 women without endometriosis the cytotoxicity against four different tumor cell lines--K562, the endometrium carcinomas AN3CA and RL95, the natural-killer (NK)-resistant Daudi cell line--was investigated, using either freshly isolated peripheral blood mononuclear cells (PBMC) or recombinant interleukin (IL)-2-stimulated PBMC. In 5 additional women collagenase-DNase-digested endometrium was used, to investigate whether recombinant IL-2-activated lymphocytes displayed an increased cytotoxicity against fresh and cultured endometrial cells. The cytotoxicity of unstimulated PBMC toward K562, AN3CA and RL95 target cells was decreased in women with endometriosis compared to women without endometriosis (p < 0.05, for all). After recombinant IL-2 stimulation the cytotoxicity toward the four different target cells increased significantly, both in women with and without endometriosis. There was no difference in LAK-mediated cytotoxicity against the four tumoral cells between women with and without endometriosis. Significant LAK activity was demonstrated against both fresh and cultured (72 h) endometrial cells. The cytotoxicity of autologous lymphocytes against cultured endometrial cells was 31.0 +/- 17 versus 67.4 +/- 5.8%, using lymphocytes cultured in medium without and with recombinant IL-2, respectively (paired t test, p < 0.02).(ABSTRACT TRUNCATED AT 250 WORDS)

Flow cytometry analysis of lymphocyte subpopulations in peritoneal fluid of women with endometriosis.

PROBLEM: We investigated the lymphocyte subpopulations in peripheral blood (PB) and peritoneal fluid (PF) of women with and without endometriosis to evaluate if the decreased natural killer (NK)-mediated cytotoxicity in women with endometriosis was due to a quantitative defect or not. METHOD: The PB and PF mononuclear cells of 59 women undergoing a diagnostic laparoscopy for pain and/or infertility were analyzed by flow cytometry. RESULTS: The number and concentration of PF mononuclear cells (MC) was increased in women with endometriosis compared to women without endometriosis. The monocyte/macrophage marker (CD14) was expressed on 70.3 and 66.9% of PFMC of women with and without endometriosis, respectively. The CD4/CD8 ratio was inverted in the PF, and this was more pronounced in women with endometriosis. In the PF of women with endometriosis, 41.3% of the lymphocytes were CD8 positive, compared to 34.3% in women without endometriosis. The percentage of NK positive lymphocytes in PF, using three different monoclonal antibodies directed against NK cell markers (CD57, CD16, and CD56) were not different between women with and without endometriosis. In women with endometriosis, 12.7, 9.5, and 28.8% of lymphocytes were CD57, CD16, and CD56 positive, respectively. CONCLUSION: PFMC consisted mainly of phagocytic and human leukocyte antigen (HLA)-restricted or HLA unrestricted cytotoxic cells capable of reacting to various antigens entering the cavity from the lower genital tractus. Furthermore, the decreased NK activity reported in PB and PF of women with endometriosis was not likely to be caused by a quantitative defect, since the percentage of NK positive lymphocytes was not different between women with and without endometriosis.

Immunosuppressive activity of peritoneal fluid in women with endometriosis.

OBJECTIVE: To investigate the immunosuppressive effect of peritoneal fluid and follicular fluid on both natural killer-mediated cytotoxicity and phytohemagglutinin-induced lymphocyte proliferation. METHODS: The peritoneal fluid of women with endometriosis was compared to both fertile and infertile control fluids. Lymphocytes were pretreated for 2 or 20 hours with peritoneal or follicular fluids, and their cytotoxicity toward K562 tumor cells was measured. We also investigated the phytohemagglutinin-induced stimulation of lymphocytes cocultured with peritoneal or follicular fluid. RESULTS: Peritoneal fluid from women with endometriosis had a significantly greater immunosuppressive effect on natural killer-mediated cytotoxicity and on phytohemagglutinin stimulation of lymphocytes compared to peritoneal fluid of fertile women without endometriosis (P < .01 and P < .05, respectively). Using the peritoneal fluid of infertile women without endometriosis, these differences were significant only when compared to women with severe endometriosis. Inhibition of the natural killer activity increased when the incubation period was prolonged from 2 to 20 hours (P < .04). There was no correlation between the immunosuppressive effect of peritoneal fluid and the volume of peritoneal fluid, the day of the menstrual period, or estradiol, progesterone, prostaglandin E2, or prostaglandin F2 alpha levels. In peritoneal fluid, the factor responsible for inhibition of natural killer activity was not removed with charcoal treatment. In follicular fluid, on the other hand, the inhibition of natural killer activity decreased significantly after treatment with charcoal. CONCLUSION: Natural killer activity is suppressed by the peritoneal fluid of women with severe endometriosis; this may be important in the pathogenesis of endometriosis. The factor responsible for the inhibition of natural killer activity in peritoneal fluid is different from that in follicular fluid.

Angiogenic activity of peritoneal fluid from women with endometriosis.

OBJECTIVE: To investigate the presence of angiogenic factors in peritoneal fluid (PF) and follicular fluid (FF). DESIGN: The PF samples of 48 women with (n = 24) or without (n = 24) endometriosis were investigated. Angiogenesis was assayed using the chorioallantoic membrane of 11-day-old fertilized chicken embryos. Glass fiber prefilters impregnated with the fluids were placed on the chorioallantoic membrane. MAIN OUTCOME MEASURE: The vascular reaction was analyzed by an independent observer after 72 hours. RESULTS: There was a positive reaction in 58.3% of the PF from women with endometriosis and in 12.5% of the women without endometriosis. No correlation was found between the angiogenic response and the severity of endometriosis. The reaction remained after charcoal treatment of the PF. Positive reaction was found in three of six FF samples. CONCLUSION: The PF of women with endometriosis contain more angiogenic factors than PF from women without endometriosis. This angiogenic activity could be important for the further outgrowth and progression of the lesions.

Immunohistochemical characterization of leucocyte subpopulations in endometriotic lesions.

Leucocyte subpopulations localized in endometriotic lesions were analysed using the avidin-biotin immunoperoxidase technique on 15 biopsies obtained by CO2 laser excision. Qualitative assessment of the leucocyte subpopulations was performed with a panel of antihuman monoclonal antibodies for leucocytes (anti-Hle-1), T-lymphocytes (anti-leu-4), T helper/inducer (anti-leu-3a), T suppressor/cytotoxic (anti-leu-2a), B cells (anti-leu-12), HLA-DR (anti-HLA-DR), macrophages (anti-leu-M3) and natural killer cells (anti-leu-7, anti-leu-11; anti-leu-19). Leucocyte common antigen (anti-Hle-1)-positive cells were present in all lesions and were the most frequent stromal leucocytes. Of these, the T lymphocytes are the most frequent subpopulation together with the macrophages. The CD4/CD8 ratio was 0.78. No anti-leu-7 and/or anti-leu-11-positive cells were found although a substantial amount of anti-leu-19-positive cells were found in each lesion. There were very few B cells present in the ectopic endometrial lesions. In conclusion, an important amount of cytotoxic lymphocytes (anti-leu-2a -and anti-leu-19-positive cells) and macrophages (anti-leu-M3) were found in the endometriotic lesions. The possible importance of these intraendometriotic leucocytes for the pathophysiology of endometriosis will be discussed.

The natural killer activity of peritoneal fluid lymphocytes is decreased in women with endometriosis.

OBJECTIVE: To investigate the local natural killer (NK) activity of the peritoneal fluid mononuclear cells (PFMC). DESIGN PATIENTS: In a prospective way, the NK activity (K562-assay) was measured in peripheral blood (PB) and peritoneal fluid (PF) of 44 women who underwent a laparoscopy for infertility and/or pain at the University Hospital of Gasthuisberg, Leuven, Belgium. MAIN OUTCOME MEASURE: The NK activity of peripheral blood mononuclear cells and PFMC, the number and concentration of PFMC, the percentage of lymphocytes versus macrophages by May-Grünwald-Giemsa staining and the estradiol and progesterone concentration of the PF were correlated together and with the severity of endometriosis. RESULTS: We demonstrated that there is a significant NK activity in PF and that this activity is decreased in women with endometriosis. This defect was more pronounced in the follicular phase of the cycle compared with the postovulatory phase. In PB of the same 44 women, the decreased NK activity correlated with the severity of the disease. This confirms our previous report on another 34 women. CONCLUSIONS: The NK activity is decreased in women with endometriosis and correlated significantly with the severity of the disease in both the PB and PF of women with endometriosis.

Women with endometriosis show a defect in natural killer activity resulting in a decreased cytotoxicity to autologous endometrium.

OBJECTIVE: The role of natural killer (NK) cells in the decreased cellular immunity of women with endometriosis was investigated. DESIGN, SETTING, PATIENTS: Thirty-four women were investigated prospectively before a CO2-laser laparoscopy for infertility and/or pain at the University Hospital Gasthuisberg. Endometriosis was scored blindly. MAIN OUTCOME MEASURE: The cytotoxicity, directed against the endometrium, was mediated by NK cells because this cytotoxicity could be removed by treating the effector cells with the NK-specific anti-Leu-11b monoclonal antibody. Consequently, we evaluated prospectively in those women the lymphocyte-mediated cytotoxicity toward NK sensitive (K562-assay) and autologous endometrial target cells. RESULTS: The NK activity (K562-assay) and the cytotoxicity against autologous endometrial cells were similarly decreased in women with endometriosis and correlated with the severity of the disease. Using heterologous effector cells, the decreased chromium release in women with endometriosis was less pronounced but still present. CONCLUSION: The decreased cytotoxicity to endometrial cells in women with endometriosis is mainly because of a defect in NK activity but is also partially because of a resistance of the endometrium to NK cytotoxicity.