{"paper_id":"05fd095f-3ed8-431b-8067-5320e553e5d4","body_text":"160 Central European Journal of Immunology 2007; 32(3)\nClinical immunology\nThe estimation of IL-2 and IL-2 receptors  \nin peritoneal fluid of infertile patients  \nwith endometriosis\nMAREK GOGACZ1, DOROTA DARMOCHWAL-KOLARZ2, LECHOSŁAW PUTOWSKI1,  \nBOGDAN KOLARZ3, JAROSŁAW CYBULSKI4, JAN KOTARSKI5\n12nd Departament of Gynecology, Medical University of Lublin, Poland; 2Department of Obstetrics and Perinatology, Medical University of \nLublin, Poland; 3Department of Reumathology and Connective Tissue Diseases, Medical University of Lublin, Poland; 4District Hospital, \nKrasnystaw, Poland; 51st Departament of Gynecology, Medical University of Lublin, Poland\nCorrespondence: Dorota Darmochwal-Kolarz, MD, PhD, Department of Obstetrics and Perinatology, Medical University of Lublin, 20-950 \nLublin, Jaczewskiego 8, Poland. Phone number: +48 81 463 26 80, fax number: +48 81 463 26 81, Email: dorotak@mp.pl\nAbstract\nBackground: The aim of the study was to investigate the concentrations of interleukin-2 (IL-2), \nsoluble interleukin-2 receptors (sIL-2R) and the expressions of CD25 antigen (IL-2R α) on T CD3 + \nlymphocytes in peritoneal fluid of infertile patients with endometrisis in comparison with the refe -\nrence group of patients with benign ovarian tumors.\nMaterial and Methods: Twenty four infertile patients with endometriosis and 18 patients from \nreference group were included in the study. The concentrations of IL-2 and soluble IL-2 receptors \nwere measured with the use of ELISA immunoenzymatic method. The expressions of surface mole -\ncules were estimated using the two-color flow cytometric method. \nResults: The concentrations of IL-2 in peritoneal fluid of infertile patients with endometriosis \nwere significantly higher when compared to the reference group (2.83±2.6 pg/ml vs. 1.27±1.58 pg/ml;  \np<0.05). The concentrations of soluble IL-2 receptors and the expressions of CD25 antigen on  \nT CD3+ lymphocytes in the study group did not differ significantly when compared to the referen -\nce group (sIL-2R: 463.9±200.4 U/ml vs. 461.35±209.7 U/ml; NS; T CD3 +CD25+: 6.77±8.18% vs. \n5.7±6.43%; NS). However, we have found significant differences in immunological parameters be -\ntween individual stages of endometriosis. The expressions of CD25 antigen were significantly higher \nin the second and third stages of endometriosis when compared to the first stage of the disease. \nConclusions:  The excessive concentrations of IL-2 in peritoneal fluid of patients with endome -\ntriosis may be responsible for an infertilty in these group of patients. Higher expressions of CD25 \nantigen on T CD3 + lymphocytes which are observed in the third and second stage of endometriosis \ncan suggest the acivation of T CD3 + cells during the progression of the disease.\nKey words: CD3+25+ T cells, endometriosis, infertility, IL-2, soluble IL-2 receptors.\n(Centr Eur J Immunol 2007; 32 (3): 160-163)\nIntroduction\nEndometriosis is characterized by the presence and \ngrowth of endometrial tissue in the locations other than \nuterine cavity. The disease affects about 10-20% of wo -\nmen at reproductive age and up to 45% of infertile pa -\ntients. Despite many efforts of the investigators to find \nfactors which cause the disease, the etiopathology of this \ndisorder remains unknown. Recent data suggest that aber-\nrant immunologic mechanisms are involved in its patho -\nphysiology. There is a general concept that endometriosis \nis a local pelvic inflammatory process with altered func -\ntion of immune cells in the peritoneal environment. Many \nstudies support the hypothesis that endometriosis is asso -\nciated with an activation of peritoneal macrophages and \nhigher concentrations of these cells in peritoneal fluid. In \n\nCentral European Journal of Immunology 2007; 32(3) 161\nThe estimation of IL-2 and IL-2 receptors in peritoneal fluid of infertile patients with endometriosis\nfact, peritoneal cavity of patients with endometriosis con-\ntains a fluid with a large amount of activated macrophages. \nThe secretory activity of these cells is increased and it is \nsuggested that they play an important role in the develop -\nment and maintenance of endometriosis [1]. The role of \nmacrophages in the pathogenesis of endometriosis is well \nproved, but the engagement of T cells in the pathogenesis \nof the disease has not been yet established. There are the \nconcepts that the secretion of Th1 and Th2 type cytokines \nis altered in women with endometriosis, suggesting that T \nhelper subsets play a role in the immunological responses \nto endometriosis [2, 3]. Some results suggest that the cyto-\ntoxic activity of NK and LAK cells is decreased in patients \nwith endometriosis [4, 5].\nThe aim of our study was estimate the concentrations \nof Interleukin-2 (IL-2), soluble IL-2 receptors (sIL-2R) and \nthe expressions of CD 25 antigen (IL-2Rα) on T CD3+ lym-\nphocytes in peritoneal fluid of infertile patients with endo-\nmetriosis and patients with benign ovarian tumors.\nMaterial and Methods\nPatients\nThe patients participating in the study were admitted to \nthe Department of Gynecology, Skubiszewski Medical Uni-\nversity of Lublin. We examined forty two patients. Twenty \nfour patients with endometriosis comprised the study group. \nA control group could not be selected because a sterilisa -\ntion is forbidden by law in Poland. Therefore we composed  \na refference group from women with benign ovarian tu -\nmors. The patients included in the reference group had sero-\nus ovarian cystadenoma. The diameters of the ovarian cysts \nwere between 3 to 6 cm. The patients from the reference \ngroup did not have the signs of inflammation in the perito-\nneal cavity. We took a peritoneal biopsy in the controls to \nrule out endometriosis.\nThe patients included in the study group had endome-\ntriosis and a primary infertility. The physical examination \nand a laboratory evaluation were performed. The diagnostic \ntests have been performed such as: thyroid stimulating hor-\nmone (TSH), thyroxine (T4), triiodothyronine (T3), prolactin \n(PRL) levels and liver function tests. All patients had nor-\nmal values of TSH, thyroid hormones, prolactin and liver \nfunction tests. All patients underwent hysterosalpinography \nbefore laparoscopy. All couples also had peripheral blood \nchromosome assessment using standard techniques. In ad-\ndition, patients with infections (viral and bacterial), hormo-\nnal abnormalities or autoimmune diseases were excluded. \nThey did not have a hormonal therapy in the last three mon-\nths preceding the laparoscopy. \nAll procedures were performed between seventh and \nninth day of the menstrual cycle with ovulation confirmed \nby midphase luteal progesterone concentrations. The study \ndesign was accepted by the local Ethics Committee. Infor-\nmed consent from the patients for peritoneal fluid sampling \nwas obtained. Patients with endometriosis were classified \naccording to the Committee on Terminology of the Ameri-\ncan Fertility Society. \nPeritoneal fluid sampling and cell preparation \nPeritoneal fluid samples from patients with endometrio-\nsis were taken during laparoscopy. Ten milliliters of perito-\nneal fluid were taken from each patient and every woman \nfrom the reference group and collected in sterile heparini-\nzed tubes. Peritoneal fluid samples were isolated using the \nlymphocyte separation medium – Lymphoprep (Nycomed, \nTorshov, Norway). Mononuclear cells were centrifuged for \n30 min at 600 g at 4°C, collected from the interface with  \na Pasteur pipette and washed twice in 2 ml of PBS by centri-\nfugation for 5 min at 250 g at 4°C. After washing, the cells \nwere resuspended in PBS with 2% FCS (Fetal Calf Serum, \nGibcoBRL, UK) and the total number of cells was deter -\nmined using the microscope. The cells were counted under \nthe microscope with the use of the chamber of Neubauer. \nThe cell density was adjusted to 107 cell/ml in PBS with 2% \nFCS. Next, the cells were labelled by direct staining with \nmonoclonal antibodies. One hundred microlitres of the cell \nsuspension were added to 10 µl of appropriate solution of \nfluorescein isothiocyanate (FITC) and phycoerythrin (PE) – \nconjugated antibodies (Becton Dickinson) in the following \ncombinations: 1. negative control – anti-IgG 1 (FITC) and \nanti-IgG2a (PE); 2. anti-CD 45 (FITC) and anti-CD14 (PE); \n3. anti-CD3 (FITC) and anti-CD25 (PE). The cell and anti-\nbody mixture was incubated for 30 min at 4°C in the dark, \nit was centrifuged, washed twice by adding 1 ml of cold \nPBS to each tube with 1% sodium azide and 1% FCS and \ncentrifuged again at 400 g for 10 min. Next, the supernatant \nwas separated. Before a cytometric analysis, each sample \nwas resuspended in 200 µl of PBS.\nFlow cytometric analysis\nThe cell phenotype characterization was performed \nusing the two-colour flow cytometric method (cytometer \n– Facs Calibur Becton Dickinson with an argon laser opera-\nting at 488 nm, equipped with analysis software-Immuno-\ncount II). The results were presented as percentage of cells \nstained with the antibody. The percentages of cells that \nwere antibody positive were calculated by comparison with \nthe appropriate control. \nThe estimation of IL-2 and sIL-2R concentrations\nSandwich enzyme immunoassay test kits were used \nfor the quantitative determination of IL-2 (Endogen Hu -\nman IL-2 ELISA kit, ENDOGEN Inc, MA, USA) and so-\nluble receptors for IL-2 Endogen Human IL-2 ELISA kit,  \nENDOGEN Inc, MA, USA).\nFor Endogen Human IL-2 ELISA kit the standard curve \nwas 0 – 1500 pg/ml, the detection limit – 6 pg/ml, inter – as-\nsay and intra – assay variation were less than 10%. \n\n162 Central European Journal of Immunology 2007; 32(3)\nMarek Gogacz et al.\nAssays for IL-2 and soluble IL-2 receptors were perfor-\nmed in the following manner – similar for each cytokine \nELISA kit. The assays were performed on the microwell \nstrips. Examined samples and the biotinylated antibody re-\nagent were pipetted into the wells coated by anti-cytokine \nantibody (or anti-soluble IL-2 receptor antibody) and the \nplate was incubated in room temperature for 3 hours. Next, \nthe plate was washed three times with wash solution, the \nstreptavidin-HRP conjugate was added to each well and \nthen the plate was again incubated at 20-25°C for 30 min. \nAfter washing, the substrate solution was pipetted into each \nwell and the plate incubated under exclusion of light. The \nreaction was terminated by addition of the stop solution. Ad-\nsorbance was read at 450 nm using ELISA reader. All deter-\nminations were performed in duplicate. Cytokine (or soluble \nIL-2 receptor) concentrations were read from the standard \ncalibration curve. The concentrations of IL-2 were presen-\nted in pg/ml and the concentrations of sIL-2R in U/ml.\nStatistical analysis\nStatistical differences between the groups were estima-\nted using a standard non-parametric test (Mann-Whitney \nU test). The results of IL-2, sIL-2R concentrations and the \nexpressions of CD25 anitgen on T CD3 + cells were pre -\nsented as the mean with SD and ranges. The correlations \nbetween different parameters were analyzed by Spearman \nrank correlation test. Differences at p<0.05 were considered \nas statistically significant.\nResults\nThe concentrations of IL-2 in peritoneal fluid of infer-\ntile patients with endometriosis were significantly higher \nwhen compared to the reference group (IL-2: 2.83±2.6 \npg/ml vs. 1.27±1.58 pg/ml; p<0.05). The concentrations \nof soluble IL-2 receptors in peritoneal fluid of patients \nwith endometriosis did not differ significantly when com-\npared to a reference group (sIL-2R: 463.9±200.4 u/ml vs. \n461.35±209.7 u/ml; NS). The expressions of CD 25 antigen \non T CD3+ lymphocytes cells in peritoneal fluid of the study \ngroup did not differ in comparison with the reference gro-\nup (T CD3+CD25+: 6.77±8.18% vs. 5.7±6.43%; NS). But \nwe have found significant differences between individual \nstages of endometriosis. The expressions of CD25 antigen \non T CD3 + cells in the third stage of endometriosis were \nsignificantly higher when compared to first stage of the di-\nsease. Similarly, the expressions of CD25 antigen in second \nstage of endometriosis were significantly higher in compa-\nrison with the first stage of the disease. The concentrations \nof IL-2, sIL-2R and the expressions of CD25 antigen on \nTCD3+ lymphocytes in perotoneal fluid in the group of pa-\ntients with endometriosis depending on stage of the disease \nare presented in table 1.\nDiscussion of results\nMany investigators observed decreased content of cy-\ntokines which reflect the function of T and NK cells, such \nas IL-2 and IFN-γ in peritoneal fluid of patients with endo-\nmetriosis [6, 7]. \nThe results of Hernandez-Guerrero at al. showed smal-\nler intracellular synthesis of IL-2 and IFN- γ in peritoneal \nfluid (PF) and peripheral blood (PB) of patients with endo-\nmetriosis. The decrease was associated with smaller percen-\ntage of activated, cooperative T lymphocytes and NK cells \nin PF of patients with endometriosis. This phenomenon was \nmore stressed at the III and IV pathology degree. The stu-\ndies of Polish group confirmed that the concentrations of \nIFN-γ were significantly decreased in the group of patients \nwith endometriosis [8].\nHsu et al. did not observed the differences in mRNA \nor protein level of IL-2 and IL-10 in PF and PB of patients \nwith endometriosis. The levels of IL-4 in PF and PB of \npatients with endometriosis were significantly higher than \nthose of normal patients [4]. \nWe observed the increased concentrations of IL-2 in pe-\nritoneal fluid of infertile patients with endometriosis. Simi-\nlar results were obtained by Chinese researchers who have \nfound the increased content of IL-2 in the cases of infertile \npatients with endometriosis [2]. \nIn contrast to these results, Ho et al. observed the decre-\nased concentrations of IL-2 in patients with endometriosis \n[9]. IL-2 is well known Th1 type cytokine responsible for \ninfertility as direct embriotoxic agent. The results of our \nstudy suggest that endometriosis complicated by infertility \nis associated with the Th1/Th2 imbalance with predomi -\nnance of Th1 type immunity. It seems possible that incre-\nased concentrations of IL-2 are the main factors which are \nTable 1. The concentrations of IL-2, sIL-2R and the expressions of CD25 antigen on T CD3+ lymphocytes in peritoneal fluid of \npatients with endometriosis depending on stage of the disease\nIL-2 (pg/ml) sIL-2R (U/ml) CD3+25+ (%)\nI stage (n=10) 2.01±2.92 598.33±212.52 1.38±1.02 \nII stage (n=8) 3.77±2.69 361.06±145.65 6.00±2.68\nIII stage (n=6) 1.97±1.41 450.73±194.25 12.14±10.83\np II vs. I (NS)\nIII vs. II (NS)\nII vs. I (p<0.05)\nIII vs. II (NS)\nII vs. I (p<0.05)\nIII vs. I (p<0.01)\nn – number of patients; p – statistical significance.\n\nCentral European Journal of Immunology 2007; 32(3) 163\nThe estimation of IL-2 and IL-2 receptors in peritoneal fluid of infertile patients with endometriosis\nresponsible for the infertility in the group of patients with \nendometriosis. \nIn our study we did not find the differences between the \nconcentrations of soluble IL-2 receptors (sIL-2R) between \nthe group of infertile patients with endometriosis and refe-\nrence group. The concentrations of sIL-2R in the first stage \nof endometriosis were significantly higher when compared \nto second stage of the disease. In the literature we did not \nfind the information about the concentrations of sIL-2 re -\nceptors in peritoneal fluid of infertile patients with endo -\nmetriosis. Koumantakis et al. estimated serum concentra -\ntions of sIL-2R. They have found that the concentrations of  \nsIL-2R were significantly higher in women with endome-\ntriosis when compared to controls [10]. \nThey have made similar observations in the three year \nfollow-up of a patient with severe endometriosis. They ob-\nserved increased serum concentrations of sIL-2R before \ntreatment which have been suppressed after Danazol admi-\nnistration [11]. \nThe percentage of T CD3+25+ cells did not differ signi-\nficantly in the group of infertile patients with endometriosis \nwhen compared to the reference group. But we observed si-\ngnificant differences between individual stages of endome-\ntriosis. The numbers of T lymphocytes with the expression \nof CD25 antigen were significantly higher in the third stage \nof endometriosis when compared to first stage of the dise-\nase. Similarly, the levels of T CD3+25+ cells in the second \nstage of endometriosis were significantly higher in compa-\nrison with the first stage of the disease. \nSotnikova et al. observed that endometriosis was as -\nsociated with high levels of expressions of CD25 and  \nHLA-DR molecules and diminished expressions of Fas \nantigen on peritoneal T CD3 + lymphocytes. Furthermore, \nthe supernatant of 24h culture of peritoneal macrophages \nfrom women with endometriosis induced the expressions \nof CD25 antigen on T CD4+ but not CD8+ lymphocytes and \ndecreased Fas expressions by both cell subsets [12].\nThe observed increase in T CD25 +3+ cells in associa -\ntion with the disease severity may reflect an increase in the \nnumber and activation of T regulatory (Treg) cells in the \nperitoneal cavity. \nReferences\n1. Keenan JA, Chen TT, Chadwell NL et al. (1995): IL-1 beta, \nTNF-alpha, and IL-2 in peritoneal fluid and macrophage con-\nditioned media of women with endometriosis. Am J Reprod \nImmunol 34: 381-385.\n2. Zhang S, Wang H, Meng C (1998): Alteration of peritoneal \nlymphocyte transformation and its interleukin-2 release in pa-\ntients with infertility and endometriosis. Zhonghua Fu Chan Ke \nZa Zhi 33: 17-19.\n3. Hsu CC, Yang BC, Wu MH, Huang KE (1997): Enhanced \nInterleukin-4 expression in patients with endometriosis. Fertil \nSteril 67: 1059-1064.\n4. Garzetti GG, Ciavattini A, Provinciali M et al. (1995): Natural \nkiller activity in stage III and IV endometriosis: impaired cy -\ntotoxicity and retained lymphokine responsiveness of natural \nkiller cells. Gynecol Endocrinol 9: 125-130.\n5. Oosterlynck DJ, Lacquet FA, Waer M, Koninckx PR (1994): \nLymphokine-activated killer activity in women with endome-\ntriosis. Gynecol Obstet Invest 37: 185-190.\n6. Wu MY, Ho HN (2003): The role of cytokines in endometrio-\nsis. Am J Reprod Immunol 49: 285-296.\n7. Hernandez Guerrero CA, Tlapanco Barba R, Ramos Perez C et \nal. (2003): Endometriosis and discouragement of immunology \ncytotoxic characteristics. Ginecol Obstet Mex 71: 559-574.\n8. Szyllo K, Tchorzewski H, Banasik M et al. (2003): The invo-\nlvement of T lymphocytes in the pathogenesis of endometriotic \ntissues overgrowth in women with endometriosis. Mediators \nInflamm 12: 131-138.\n9. Ho HN, Chao KH, Chen HF et al. (1995): Peritoneal natural \nkiller cytotoxicity and CD25+CD3+ lymphocyte subpopula -\ntion decreased in women with stage III-IV endometriosis. Hum \nReprod 10: 2671-2675.\n10. Koumantakis EE, Matalliotakis IM, Neonaki M et al. (1994): \nSoluble serum Interlukin-2 receptor, Interlukin-6 and Interlu-\nkin-1α in patients with endometriosis and in controls. Arch \nGynecol Obstet 255: 107-112.\n11. Matalliotakis IM, Neonaki M, Panidis DK et al. (2000): Three-\n-year follow-up of [CA-125, CA 19-9, CA 15-3, SIL-2R, IL-6, \nIL-la, TNF-a, sCD8 and sCD4] levels in a woman with severe \nendometriosis. Eur J Obstet Gynecol Reprod Biol 93: 127 -\n-129.\n12. Sotnikova NY, Antsiferova YS, Posiseeva LV et al. (2000):  \nA Study of Peritoneal Immunocompetent Cells in External Ge-\nnital Endometriosis. Russ J Immunol 5: 307-314.","source_license":"CC0","license_restricted":false}