Mark Waer

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.

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.

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.

Immunohistochemical characterization of leucocyte subpopulations in endometriotic lesions

SummaryLeucocyte 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 intra-endometriotic leucocytes for the pathophysiology of endometriosis will be discussed.

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)

Immunologic changes after loco-regional radiotherapy and fractionated total body irradiation (TBI) in mice

The immunologic effects of fractionated irradiation to both hind limbs and the tail of adult (2.5-3 months old) male Balb/c mice were investigated. A dose of 34 Gy given in 17 fractions of 2 Gy, 1 fraction per day, 5 days per week, was delivered with a 60Co source. A significant decrease of the total splenocyte count (29% of control value) and of the PHA(phytohemagglutinin)-induced proliferation of T cells (22% of control value) was found immediately after irradiation. Both parameters normalized within 30 days after irradiation. Immediately after irradiation, the MLC (mixed lymphocyte culture) was supranormal (126% of control value), dropped to 45% 1 week later, and normalized within 1 month after radiotherapy. The NK (natural killer) activity was significantly decreased only the first week after loco-regional irradiation, while the LAK (lymphokine activated killer) activity was not altered at all. The percentage of goat-anti-mouse+ cells (mainly B lymphocytes) was not changed immediately after loco-regional irradiation, but rose to supranormal values (175% of control level) 3 months after irradiation. A persistent decrease of the percentage and the absolute numbers of the Lyt2+ cells (= CD8+ cells, suppressor/cytotoxic phenotype) was observed up to 3 months after irradiation, while the percentage of L3T4+ cells (= CD4+ cells, helper phenotype) remained normal for the total follow-up. No differences in allogeneic skin graft survival could be demonstrated between irradiated and control animals. The observed immunological effects could not be explained by the scatter irradiation to the whole body as total body irradiation (TBI) administered in a dose and dose rate similar to the scatter dose did not result in persistent immunologic changes. No dose-rate effect could be demonstrated in a low dose fractionated total body irradiation schedule. A total body irradiation similar to the scatter dose in humans did not result in significant immunologic changes.

Influence of overall treatment time in a fractionated total lymphoid irradiation as an immunosuppressive therapy in allogeneic bone marrow transplantation in mice

Three groups of C57/BL/Ka mice received total lymphoid irradiation (TLI) in a total dose of 34 Gy in three different fractionation schedules. In the first group daily fractions of 2 Gy were given during 3 1/2 weeks. In the second group 4 to 5 fractions with 3 1/2 hr interval were given each day, thus delivering 17 fractions in 4 days. In the third group three fractions were given daily for two consecutive days and was repeated two times after 8 or 9 days interval, resulting in a total treatment time of 3 1/2 weeks. The tolerance of all different schedules was excellent. No difference in the peripheral white blood cell and lymphocyte counts nor the degree of immunosuppression as measured by phytohaemaglutinin or concanavalin A induced blastogenesis and mixed lymphocyte reaction were observed at the end of the treatment and up to 200 days. When bone marrow transplantation was performed one day after the end of each schedule, chimerism without signs of graft versus host disease was induced in all the groups. However, from the results in a limited number of animals it seems that concentrated schedules were less effective for chimerism induction. It has been demonstrated that it is possible to reduce drastically the overall treatment time for TLI before bone marrow transplantation. Further investigations are necessary in order to determine the optimal time-dose-fractionation factors and the different parameters involved in the transplantation.

Normal and Malignant Trophoblasts Do Not Recruit Granulated Metrial Gland Cells

A possible relationship between the development of granulated metrial gland (GMG) cells and trophoblast was studied. Trophoblast implanted in ectopic sites (e.g. kidney capsule) did not induce decidua and did not recruit GMG cells. Only when injected in utero did trophoblast lead to the development of decidua and to the recruitment of GMG cells. With malignant trophoblast (choriocarcinoma cells) similar results were obtained as with normal trophoblast both after ectopic or after in utero injection. The presence of decidua, but not the development of a conceptus or the outgrowth of trophoblast, seems to be required for the differentiation of GMG cells.

Mechanisms involved in the differential recovery of CD4 and CD8 T-lymphocytes after local irradiation in mice

The mechanisms involved in the differential recovery of CD4 (helper/inducer phenotype) and CD8 (cytotoxic/suppressor phenotype) T-lymphocytes after fractionated local irradiation were investigated. In mice, a better recovery of CD4 cells than of CD8 cells was found, while the reverse has been described in humans. Differences in radiosensitivity between CD4 and CD8 mouse splenocytes could not be found. No sequestration of CD8 cells in irradiated tissues could be demonstrated. Irradiation of the thymus did not influence the observed immune changes. Altered thymic production of CD4 and CD8 cells could be excluded by intrathymic injection of FITC (fluorescein isothiocyanate). Hindlimb and tail irradiation did not induce changes in the morphology of the thymus and the phenotype of the thymocytes. These results suggest that the differential recovery of CD4 and CD8 T-lymphocytes after local irradiation is determined by extrathymic factors in man and mice, and that the observed differences in immune recovery between man and mice are due to defective thymic function in the former and normal function in the latter.

Determination of mixed chimerism by a simple flow cytometry method

A simple, sensitive and accurate method was developed to determine the level of lymphoid chimerism in bone marrow-transplanted rodents. The method is based on flow cytometry using polyclonal alloantisera and labeled second step anti-IgG antibodies. Using mixtures of spleen cells from different mouse strains, it was demonstrated that low levels of chimeric cells (less than 1%) could easily be detected. Moreover, using two-color fluorescence analysis, the level of chimerism could also be determined in subpopulations of lymphoid cells, e.g., CD4 or CD8 cells and was found to be identical to the results obtained in unseparated lymphoid populations. The method was compared to the complement dependent cytotoxicity assay (CDCA) and to the flow cytometric determination of chimerism using labeled monoclonal antibodies against specific MHC antigens. CDCA was found to be more labor intensive and could only estimate the composition of the cell mixtures without detecting low levels of chimerism (less than 5%). The results of flow cytometry, using directly labeled monoclonal antibodies or polyclonal antibodies with second step reagents, were identical. It is concluded that, due to its simplicity and high sensitivity, the method described permits reliable determination of the level of mixed chimerism in rodents and is an excellent alternative when no anti-MHC mAbs are available.