American Journal of Obstetrics and Gynecology
Volume 175 • Number 5 • November 1996
Copyright © 1996 Mosby-Year Book, Inc.

Dr.Sinan DOĞANTÜRK

Ankara

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Gynecology

Decrease in interferon gamma production and impairment of T-lymphocyte proliferation in peritoneal fluid of women with endometriosis

Hong-Nerng Ho MD

Ming-Yih Wu MD

Kuang-Han Chao MD

Chin-Der Chen MD

Shee-Uan Chen MD

Hsin-Fu Chen MD

Yu-Shih Yang MD, PhD

Taipei, Taiwan, Republic of China

Department of Obstetrics and Gynecology,
College of Medicine and the Hospital, National Taiwan University.

OBJECTIVE: Our purpose was to verify regional immune modulations and to test the effect of gonadotropin-releasing hormone agonist in women with endometriosis.

STUDY DESIGN: Concentrations of peritoneal cytokines, including interleukin-1beta, interleukin-2, soluble interleukin-2 receptor, interleukin-6, granulocyte-monocyte colony-stimulating factor, interferon gamma, and tumor necrosis factor-alpha were compared in women with and without endometriosis. Peritoneal cytokine and interleukin-2 production were examined by adding various mitogens to peritoneal fluid mononuclear cells of women with advanced endometriosis before and after gonadotropin-releasing hormone agonist treatment.

RESULTS: A significant increase in peritoneal interleukin-1beta, interleukin-6, and tumor necrosis factor-alpha and a decrease in interferon gamma were noted in women with endometriosis. After gonadotropin-releasing hormone agonist treatment interleukin-6 decreased and interferon gamma increased. A significant impairment of interleukin-2 production of peritoneal fluid mononuclear cells by phytohemagglutinin and pokeweed mitogen stimulation was demonstrated in endometriosis, and production could be restored after gonadotropin-releasing hormone agonist treatment.

CONCLUSION: These results indicate that regional immunologic dysfunction might be invoked in the disease process of endometriosis. (Am J Obstet Gynecol 1996;175:1236-41.)

Key words:

Endometriosis

interleukin-6

interferon gamma

T-cell activation

gonadotropin-releasing hormone agonist

Supported in part by grant No. NSC-85-2331-B002-112 from the National Science Council and grant No. DOH-85-TD-033 from the Department of Health, the Executive Yuan of the Republic of China.

 

Received for publication October 10, 1995; revised April 9, 1996; accepted May 14, 1996.

Reprint requests: Hong-Nerng Ho, MD, Department of Obstetrics and Gynecology, National Taiwan University Hospital, No. 7 Chung-Shan South Road, Taipei, Taiwan, 100, Republic of China.

Copyright © 1996 by Mosby -Year Book, Inc.

6/1/74925

Endometriosis is the implantation of endometrial tissue into places where it is normally not found. One pathogenetic theory suggests that a deficient immune response to endometrial cells during retrograde menstruation may be involved in the establishment of the disease. ^[1] Deficient cellular immunity, in particular decreased peritoneal natural killer cell activity, ^{[2] [3]} has been proposed as an etiologic factor that could contribute to the survival, implantation, and proliferation of these regurgitated endometrial cells. However, there was no difference in the proportion of natural killer cells in peritoneal fluid mononuclear cells in patients with endometriosis and in controls. ^{[2] [4]} It was thus assumed that the decreased natural killer cell activity in endometriosis was not caused by a quantitative but by a functional defect.

Numerous studies have characterized the lymphocyte subpopulations in normal eutopic endometrium and suggested a role for cytokine secretory products of these lymphocytes in regulating endometrial cell proliferation and differentiation. ^[5] One recent study showed that there is an increased concentration of stromal T cells and an increased expression of T-cell activation in ectopic endometrium ^[6] and indicated that cytokine products of these activated T cells might be involved in regulating cellular processes of endometriotic tissue. The existence of soluble nonspecific immunosuppressive factors released from the ectopic endometrium ^{[7] [8]} was also reported, and it was proposed that the immunosuppressive factors secreted by endometriotic tissues may be involved in the local implantation and development of endometriosis. Cytokines are increased in the peritoneal fluid of patients with endometriosis and in cultured ectopic endometrial cells. ^{[9] [10] [11] [12]} Our previous studies ^{[2] [4]} have shown the impairment of peritoneal cellular immunity in women with endometriosis through the suppression of the CD25CD3 lymphocyte subpopulation and natural killer cell cytotoxicity. To further assess whether the cytokines in the peritoneal

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fluid may affect cellular immunity and play a role in regulating cell proliferation in endometriosis, we compared various cytokines in the peritoneal fluid of women with and without endometriosis. Whether these deviations could be corrected by long-term use of gonadotropin-releasing hormone (GnRH) agonist was also examined with 6-month GnRH agonist treatment in women with advanced endometriosis.

Interleukin-2 (IL-2) is a lymphokine synthesized and secreted primarily by T helper lymphocytes that have been activated by stimulation with certain mitogens or by interaction of the T-cell receptor complex with antigen/major histocompatibility complex (MHC) on the surface of antigen-presenting cells. Natural killer cells respond to IL-2 when they are properly activated. ^[13] To test whether T-cell function is impaired, as indicated by decreases in the CD25CD3 lymphocyte subpopulation, we further measured the in vitro production of IL-2 by peritoneal fluid mononuclear cells in women with and without endometriosis by adding various mitogens (phytohemagglutinin, concanavalin A, or pokeweed mitogen).

Material and methods

Experiment 1

Subjects and specimens collection.

Peritoneal fluid was obtained from women undergoing laparoscopy for infertility and tubal ligation at the Department of Obstetrics and Gynecology, National Taiwan University Hospital, Taipei, from July 1993 to June 1995. None of these women received any hormonal treatment during the 3 months preceding the laparoscopic operation, which was performed in the early follicular phase after menstruation. Seventeen women with endometriosis, including early (stages I and II, n = 3) and advanced (stages III and IV, n = 14) stages of endometriosis were scored by the revised American Fertility Society classification. Among them, 14 in the advanced-stage group received 6 months of GnRH agonist treatment (leuprolide acetate depot [Takeda, Osaka], 3.75 mg/mo subcutaneously). They underwent second-look laparoscopy and peritoneal fluid was aspirated to perform the cytokine study. No endometriosis or any other pathologic disorder, including infection, was found in another 18 women (control group).

All peritoneal fluid was aspirated from the Douglas pouch immediately after insertion of the trocar to minimize contamination with blood. Peritoneal fluid was collected in sterile heparinized tubes at room temperature. Samples with blood contamination from obvious bleeding of the puncture sites were discarded. The peritoneal fluid specimens were transferred immediately to the laboratory and processed within 30 minutes. The peritoneal fluid was centrifuged (400 g, 10 minutes) and the supernatant was frozen at -70° C as soon as possible.

Cytokine assays.

Peritoneal fluid interleukin-1beta (IL-1beta), IL-2, soluble IL-2 receptor, interleukin-6 (IL-6), granu-locyte-macrophage colony-stimulating factor (GM-CSF), interferon gamma (IFN-gamma), and tumor necrosis factor-alpha (TNF-alpha) were measured in triplicate with commercial kits of standard enzyme-linked immunosorbent assay (ELISA). The principle of the method was a solid-phase ELISA based on the ``dual-antibody immunometric sandwich'' with two distinct antibodies to cytokines derived from two different species. The assays have their lower detection limits of 0.05 pg/ml, 2.5 pg/ml, 6.0 pg/ml, 0.7 pg/ml, 2.8 pg/ml, 0.03 IU/ml, and 0.18 pg/ml for IL-1beta, IL-2, soluble IL-2 receptor, IL-6, GM-CSF, IFN-gamma, and TNF-alpha, respectively. The kits for these cytokines were from R&D Systems (Minneapolis). All the assays were performed at the same time for each peritoneal fluid sample to prevent any changes because of freezing and thawing.

Statistical analyses.

All data of peritoneal fluid cytokines except IFN-gamma (in international units per milliliter) are shown in picograms per milliliter and expressed as mean ± SD. The levels of various cytokines in the peritoneal fluid of women with and without endometriosis were evaluated by a nonparametric method (Mann-Whitney test). The comparison between the levels of various cytokines in the peritoneal fluid of 14 women with advanced endometriosis before and after 6 months of GnRH agonist treatment were calculated by the Wilcoxon signed-rank test. Type I error shown <0.05 (i.e., p < 0.05) were considered significant.

Experiment 2

T-lymphocyte proliferative assay.

Peritoneal fluid mononuclear cells were collected and prepared in sterile RPMI 1640 growth medium (Life Technologies, Grand Island, N.Y.) at a concentration of 2 × 10 ^[6] cells/ml. The lympho-proliferative assay was set up in four-well multidish culture plates (Nunclon, Roskilde, Denmark). Aliquots of 300 mul of peritoneal fluid mononuclear cell suspension were dispensed in each well and another 300 mul of RPMI 1640 medium (control), concanavalin A (0.4 mg/ml), phyto-hemagglutinin (1 mg/ml), or pokeweed mitogen (0.5 mg/ml) was added to each well individually. All three plant mitogens were obtained from Sigma (St. Louis). Plates were incubated in a humidified atmosphere of 5% carbon dioxide at 37° C for 72 hours. The conditioned media were collected and stored at -70° C for cytokine assay.

IL-2 cytokine assay.

The IL-2 concentration of conditioned media was measured in triplicate with a standard ELISA commercial kit, as mentioned above. This kit has a lower limit of 6.0 pg/ml for our culture medium sample, with intraassay coefficients of variation <7% and interassay coefficients of variation <10%.

Statistical analyses.

Data of IL-2 concentrations are shown in picograms per milliliter and expressed as mean ± SD. The distribution of IL-2 concentrations

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*Mann-Whitney test was used to compare; p < 0.05 was considered statistically significant.

among the study patients was noted to be nonnormal, and hence these data were calculated by nonparametric methods (Mann-Whitney test for comparison between control and endometriosis groups, and Wilcoxon signed-rank test for comparison of the response before and after GnRH agonist treatment).

Results

Experiment 1.

Analysis of intergroup differences of various cytokines in peritoneal fluid of women with and without endometriosis was performed by the Mann-Whitney test because of the nonparametric nature of those data. Analysis demonstrated a significant increase in IL-1beta, IL-6, and TNF-alpha (Table I) in women with endometriosis. A significantly lower concentration of IFN-gamma in the peritoneal fluid of women with endometriosis was also observed. The levels of soluble IL-2 receptor were similar in both groups. IL-2 and GM-CSF concentrations were lower than the lowest limits of detection (2.5 and 0.7 pg/ml, respectively).

After 6 months of GnRH agonist treatment the levels of various cytokines in peritoneal fluid of 14 women with advanced endometriosis were compared with levels before treatment by use of the Wilcoxon signed-rank test. The results demonstrated a significant decrease in IL-6 and an increase in IFN-gamma (Table II) . Also concentrations of IL-1beta, soluble IL-2 receptor, and TNF-alpha decreased to the levels of the controls but were not significantly different from those before treatment ( p = 0.301, 0.735, and 0.063, respectively, Table II) . IL-2 and GM-CSF were still lower than the lowest limits of detection.

Because the SD of the data was high (Tables I and II) , it might be argued that these statistical results were skewed because of a few patients with extremely high or low levels of cytokines. Therefore a chi² analysis was used to compare the number of patients with levels 2 SD units higher or

lower than the mean levels of controls. The results were similar to those by Mann-Whitney test except for the result of peritoneal IL-1beta. The difference in the level of peritoneal IL-1beta lost statistical significance between controls and patients with endometriosis. The level of peritoneal IL-6 in women with endometriosis was significantly different from that of control women ( p = 0.0007, 10/17 vs 1/18). The difference before and after GnRH agonist treatment in women with advanced endometriosis was also significant ( p = 0.00008, 10/14 vs 0/14). The level of TNF-alpha in women with endometriosis was not significantly higher than that of controls ( p = 0.15, 4/17 vs 1/18), but the difference before and after GnRH agonist treatment in women with advanced endometriosis was significant ( p = 0.04, 4/14 vs 0/14). Because the mean level of IFN-gamma of control subjects was <2 SD, we were unable to use the same statistical analysis to test the difference in IFN-gamma between control subjects and those with endometriosis.

Experiment 2.

IL-2 production in mitogen-stimulated peritoneal fluid mononuclear cells of women with and without endometriosis is shown in Table III . Phytohemagglutinin and pokeweed mitogen increased IL-2 secretion significantly, but concanavalin A did not have the same effect. Production of IL-2 was lower by phytohemagglutinin and pokeweed mitogen stimulation in the endometriosis group ( p = 0.0001 and 0.024, respectively, compared with controls).

After 6 months of GnRH agonist treatment, peritoneal fluid mononuclear cells of the same patients with advanced endometriosis were evaluated by mitogen stimulation. The diminished phytohemagglutinin stimulation effect noted before treatment was restored to the level of the control group after long-term GnRH agonist use ( p = 0.001), and the diminished pokeweed mitogen

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*Mann-Whitney test was used to compare controls and pre-GnRH agonist -treated endometriosis group; p < 0.05 was considered statistically significant.
Comparisons were made between before and after GnRH agonist use in endometriosis group by Wilcoxon signed-rank test; a p < 0.05 was considered statistically significant.

stimulation effect before GnRH agonist treatment increased after treatment but did not reach statistical significance ( p = 0.110).

Comment

In this study we demonstrated that the levels of cytokines IL-1beta, IL-6, and TNF-alpha were elevated in the peritoneal fluid of women with endometriosis compared with controls, whereas IFN-gamma levels were lower. After GnRH agonist treatment, IL-6 decreased and IFN-gamma increased significantly.

Although IL-1 production is generally considered to be a consequence of inflammation, recent evidence suggests that IL-1 also is involved in a wide variety of biologic activities, including prostaglandin synthesis and protein synthesis, and has various effects on the central nervous system. It also plays an important role in immune functions. IL-1 acts on macrophage-monocytes, inducing its own synthesis as well as the production of tumor necrosis factor and IL-6. ^{[14] [15]} IL-1 also activates T cells, resulting in IL-2 production and expression of IL-2 receptors. ^[16] IL-1 further induces the production of GM-CSF from activated T cells. ^[17] However, in our previous studies ^{[2] [4]} the CD25CD3 lymphocyte subpopulation was suppressed in patients with endometriosis. Therefore we hypothesize that the elevations of IL-1beta, IL-6, and TNF-alpha might be due to inflammatory reaction to ectopic endometrial tissue in the peritoneal cavity and might further contribute to the progression of endometriotic lesions.

A higher level of chemotactic activity of macrophages, and factors that may contribute to macrophage recruitment and activation, have recently been found in the peritoneal fluid of patients with endometriosis. ^[9] It was hypothesized that activated peritoneal macrophages may contribute to the pathogenesis of endometriosis through secretion of cytokines and growth factors. Akoum et al. ^[11] recently demonstrated that both fibroblast-like and epithelial cells of ectopic endometrium can secrete a specific chemotactic and activating factor for monocytes (MCP-1) after stimulation with IL-1beta and TNF-alpha. As we showed in this work, elevations of IL-1beta and TNF-alpha in the peritoneal fluid have also been found in women with endometriosis. ^{[12] [18]} Therefore the higher levels of IL-1beta and TNF-alpha in the peritoneal fluid of patients with endometriosis may be due to secretion by endometriotic tissue, which may be capable of producing activating or chemotactic factors to peritoneal macrophages.

IL-6 and IFN-gamma are cytokines known to modulate various aspects of the immune response. In this study IL-6 was elevated and IFN-gamma lower in the peritoneal fluid of patients with endometriosis, and the levels of both were restored significantly after 6 months of GnRH agonist treatment. Therefore levels of these cytokines may present typical immunologic modulations in the endometriosis and reflect the posttreatment condition. IL-6 promotes cellular proliferation, whereas IFN-gamma inhibits proliferation of human endometrial epithelium. ^{[19] [20]} Elevation of IL-6 has been noted in ectopic endometrial tissue culture, ^[21] peripheral blood, ^[22] and peritoneal fluid ^[23] of women with endometriosis. The elevation of IL-6 in peritoneal fluid of women with endometriosis in our study is in agreement with that reported by Boutten et al., ^[23] who suggested that IL-6 may play a significant role in the pathogenesis of endometriosis.

IFN-gamma will be low if the T or natural killer cells are inactivated. This result further confirmed our previous reports ^{[2] [4]} that natural killer cell cytotoxicity was decreased and the activated T-lymphocyte subpopulation (CD25CD3) of peritoneal fluid mononuclear cells was decreased. Function of T and natural killer cells could be restored after long-term use of GnRH agonist, ^[4] as could the concentration of IFN-gamma. However, whether the decrease in IFN-gamma is the cause or result of the suppression of peritoneal natural killer cell cytotoxicity and whether the decrease in IFN-gamma is due to the suppression of the CD25CD3 lymphocyte subpopulation need to be further clarified.

To further test whether the T cells are suppressed in

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endometriosis, we performed a T-lymphocyte proliferative assay with various mitogens on peritoneal fluid mononuclear cells of women with and without endometriosis. Depending on the stimulus, lymphocytes involved in the proliferative response can include either T cells, B cells, or both. Phytohemagglutinin and concanavalin A preferentially stimulate certain T cells. Pokeweed mitogen is a stimulant of T cells and also of B cells, in a T-cell-dependent manner. Using IL-2 production as an indicator of T-cell activation, we noted impaired proliferative response by phytohemagglutinin and pokeweed mitogen in the peritoneal fluid mononuclear cells of women with endometriosis. In our previous study T-cell activation marker (CD25CD3) in peritoneal fluid mononuclear cells was suppressed in endometriosis, ^[2] and in this study soluble IL-2 receptor concentrations in peritoneal fluid did not increase in endometriosis. These results suggest that peritoneal T-cell inactivation occurs in endometriosis.

GnRH agonist is the only medication other than danazol that has received U.S. Food and Drug Administration approval for the treatment of endometriosis. Clinically, it has been used as effectively as danazol in the reduction of endometriotic implants. We have demonstrated some immunologic alterations in the peripheral blood of women with superovulation by use of GnRH agonist. ^[24] In peritoneal fluid mononuclear cells we have also demonstrated the restoration of impaired natural killer cell cytotoxicity and suppressed CD25CD3 subpopulation by use of GnRH agonist in patients with advanced endometriosis. ^[4] In this study we further showed restoration to control levels of the alterations of cytokines in peritoneal fluid after long-term use of GnRH agonist in women with advanced endometriosis. In addition, the decrease in mitogen-stimulated lymphocyte proliferation could also be corrected to control levels after long-term treatment. Recent studies have shown that IL-2 ^[25] and IFN-gamma ^[8] may be able to correct the immunologic defect of endometriosis and indicated the capability of restoring cytolytic activity. In this study both peritoneal fluid mononuclear cell production of IL-2 after mitogen stimulation and IFN-gamma concentrations in peritoneal fluid were restored to control levels after 6 months of GnRH agonist treatment. These results confirmed previous assumptions.

In summary, significant elevations of peritoneal IL-1beta, IL-6, and TNF-alpha were found in women with endometriosis compared with controls. Peritoneal IFN-gamma was significantly lower in women with endometriosis than in those without. After a 6-month GnRH agonist treatment, IL-6 concentrations decreased and IFN-gamma concentrations increased significantly. By phytohemagglutinin or pokeweed mitogen stimulation, a significant impairment of IL-2 production by peritoneal fluid mononuclear cells in endometriosis was noted; this impairment could be restored after GnRH agonist. Impairment of natural killer cell cytotoxicity and a decrease in IL-2 and IFN-gamma production may be involved in the development and progression of endometriosis. Studies are now underway in our laboratory to determine whether in vitro recombinant IFN-gamma can return the suppressed natural killer cell cytotoxicity in the peritoneal fluid mononuclear cells of women with endometriosis to normal levels and which lymphocyte subpopulation (possibly the CD25CD3 subpopulation) is altered and corrected throughout the disease and treatment processes.

We thank Ms. Yen-Lin Chung for her excellent technical assistance.

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