{"paper_id":"05832791-341e-4470-b222-724c2e48fe5f","body_text":"CD4+, CD8+ and CD4+CD25+ T lymphocytes \nin peripheral blood and peritoneal fluid of women \nwith endometriosis - preliminary report\nJarosław Górski1, Krzysztof Szyłło1, Małgorzata Banasik2, Przemysław Lewkowicz2, Henryk Tchórzewski2\nAbstract \nIInnttrroodduuccttiioonn::  CD4+CD25+ cells play a major role in maintenance of immunological\nself-tolerance and negative control of pathological and physiological immunological\nreactions. Their absence or ill function leads to autoimmunization, immunopatho-\nlogy and allergy.\nThe aim of this study was to investigate the prevalence of T lymphocyte subpopu-\nlations in peripheral blood and peritoneal fluid of women with endometriosis, as\nwell as IL-10 generation by peripheral blood lymphocytes.\nMMaatteerriiaall  aanndd  mmeetthhooddss::  The studied group comprised 24 women with endometriosis\ndiagnosed during laparoscopy. The studied group was divided into two parts,\ndepending on progression stage. The control group comprised 12 women without\nendometriosis. The percentages of subpopulations of T lymphocytes were evaluated\nvia flow cytometry with IMK Plus test (BD Biosciences, USA). IL-10 concentration was\nanalyzed by enzyme-linked immunosorbent assay – ELISA (R&D System, USA).\nRReessuullttss:: In the present study we observed higher percentage of CD4\n+CD25+ cells\nin peripheral blood of affected women in comparison with healthy subjects. We\ndid not observe such a difference for the peritoneal fluid between the groups.\nChanges in number of CD4\n+CD25+ affect CD8 + shift.\nWe observed a decreased amount of IL-10 in peripheral blood in women with\nendometriosis. The increase after PHA (phytohaemagglutinin) stimulation was lower\nin women suffering from endometriosis than in the control group.\nCCoonncclluussiioonnss::  The quantitative disproportion of CD4\n+CD25+ T cells between peripheral\nblood and peritoneal fluid can affect the onset and development of endometriosis.\nThe impaired migration and function of Treg cells alter composition of peritoneal\nfluid, which favours the onset and development of endometriosis. \nKKeeyy  wwoorrddss::  endometriosis, Treg cells, lymphocyte subsets, IL-10.\nIntroduction\nEndometriosis, defined as the presence of endometrial mucosa together\nwith its stroma outside the original site, is a common gynaecological disease\nobserved in 10-15% of women at reproductive age. In women with infertility\nthe prevalence is higher: 50-60% [1]. \nThe aetiology of disease development has not been elucidated. Currently,\nthe most widely accepted is the theory of retrograde menstruation with further\nimplantation of endometrial cells into the peritoneum. Some authors suggest\n1Surgical Gynaecology Department, Polish Mother’s Health Centre Research Institute,\nLodz, Poland \n2Clinical Immunology Department, Polish Mother’s Health Centre Research Institute,\nLodz, Poland \nSSuubbmmiitttteedd:: 12 December 2006\nAAcccceepptteedd:: 11 March 2007\nArch Med Sci 2007; 3, 1: 37-42\nCopyright © 2007 Termedia & Banach\n37Original paper\nCCoorrrreessppoonnddiinngg  aauutthhoorr::\nKrzysztof Szyłło, MD, PhD\nSurgical Gynaecology\nDepartment, Polish Mother’s\nHealth Centre Research Institute\n281/289 Rzgowska Street\n93-338 Lodz, Poland\nPhone: +48 42 2711516\nFax: +48 42 2711460\nE-mail: kgoczmp@poczta.onet.pl\n\n38 Arch Med Sci 1, March / 2007\nthat disorders of local immune response in the\nperitoneum favour implantation and development of\nectopic endometrium [2]. These abnormalities include\nadhesion molecule expression, the chemokine-cytokine\nnetwork, and cell response. Immune homeostasis\ndisorders can facilitate implantation, proliferation and\nangiogenesis of endometrial tissue, simultaneously\ndisturbing the reproduction process [2, 3].\nThere have been reports on the autoimmune nature\nof endometriosis, due to some criteria, characteristic\nof autoimmune diseases, that it meets: generation\nof autoantibodies (to endometrium, ovaries, phospho-\nlipids and histones) and coexistence of other auto-\nimmune diseases, e.g.: systemic lupus erythematosus,\nrheumatoid arthritis, immunologically determined\nhabitual miscarriages [3].\nThe presence of antibodies may suggest generalized\nautoimmune syndrome. It has been hypothesized that\ntheir presence could make the recognition of ectopic\nendometrium via cytotoxic and helper T cells more\ndifficult, due to receptor sites being blocked. Another\nhypothesis is that autoantibodies are generated as\na result of abnormal immune response to ectopic\nendometrium changed by inflammation.\nMatarese et al. [3] suggest that endometriosis is an\nautoimmune disease with genetic, hormonal and\nenvironmental factors having a role in its pathogenesis.\nAutoimmune diseases affect about 5% of the\ngeneral population and are more common in women.\nTheir essence is abnormal recognition of self-anti-\ngens as foreign antigens, which leads to incorrect\nand excessive response of the immune system. Only\nin recent years have the cells responsible for immune\nresponse regulation been successfully isolated.\nCD4\n+CD25+ regulatory cells were first described\nby Sakaguchi et al. in 1995. From experiments on\nmice he learned that the Treg cell population is\nresponsible for suppression of autoimmunization [4].\nThese cells suppress the proliferation and function\nof self-reactive T cells. Deficiency, absence or\nabnormal functioning of these cell populations leads\nto development of autoimmune diseases. CD4\n+CD25+\ncells play a major role in the maintenance of immune\nself-tolerance and negative control of pathologic\nimmune reactions. Their absence or dysfunction\ncould lead to autoimmunization, immunopathology\nand allergy [5, 6, 7, 8, 9]. \nThe aim of this study was to investigate the\nprevalence of T lymphocyte subpopulations in\nperipheral blood and peritoneal fluid in women with\nendometriosis, as well as IL-10 generation by\nperipheral blood lymphocytes. The number of\ndeliveries and the main disorders in the groups have\nbeen analysed additionally.\nMaterial and methods\nTThhee  ssttuuddyy  ggrroouupp\nThe studied group comprised 24 women with\nendometriosis diagnosed during laparoscopy in the\nDepartment of Gynaecological Surgery of the Polish\nMother’s Health Centre Research Institute, in the\nyears 2003-2005. The laparoscopy was performed\nas a diagnostic means in infertility, small pelvic pain\nand tumours of adnexa. In each case the in-operation\ndiagnosis was verified by histopathology. The severity\nof endometriosis was defined according to the\nimproved American Fertility Society classification. The\nstudied group was divided into two parts, depending\non progression stage of the disease – I/II° (12 women)\nand III/IV° (12 women). The control group comprised\n12 female patients without endometriosis as observed\nin laparoscopy. Age range of the patients treated for\nendometriosis was between 22 and 52 years old with\nthe average age 34 years old. Accordingly in the\ncontrol group the age ranged from 17 to 47 years old\n– average 36 years old. Among women with\nendometriosis 91.7% inhabited urban areas, whereas\n8.3% inhabited rural areas. In the control group\nrespectively 83.4% and 16.6%.\nPeripheral blood was obtained before surgery with\nlithium heparin as an anticoagulant; peritoneal fluid\nwas collected during laparoscopy into sterile apyro-\ngenic test tubes. \nWritten, informed consent from each patient was\nobtained before the study.\nThe study was approved by the Polish Mother’s\nHealth Centre Research Institute Bioethical Committee\nand it was financed with the Department’s own funds.\nAAnnaallyyssiiss  ooff  ppeerriipphheerraall  bblloooodd  aanndd  ppeerriittoonneeaall\nfflluuiidd  llyymmpphhooccyyttee  ppooppuullaattiioonnss\nThe percentages of CD4 +, CD8+ and CD4 +CD25+\nsubpopulations of T lymphocytes were evaluated via\nflow cytometry with IMK Plus test (BD Biosciences,\nUSA) and using anti-CD4 (FITC, SK3 clone) and\nanti-CD25 (PE, 2A3 clone) monoclonal antibodies (BD\nPharmingen, USA), in peripheral blood and peritoneal\nfluid. 100 μL of blood or peritoneal fluid cells\nsuspension was incubated at room temperature\ntogether with the appropriate amount of monoclonal\nantibodies. In evaluation, flow cytometer FACSCalibur\nwith 488 nmm argon laser (BD Biosciences, USA)\nwas used. To analyze the results, SimulSET and\nCellQuest software (BD Biosciences, USA) was\napplied. The results are given as percentages of\ndouble-positive cells in an analyzed sample.\nAAnnaallyyssiiss  ooff  IILL--1100  pprroodduuccttiioonn  bbyy  ppeerriipphheerraall\nbblloooodd  mmoonnoonnuucclleeaarr  cceellllss  ((PPBBMMCC))\nIL-10 production level was analyzed in supernatants\nobtained after 72 hours of incubation of isolated\nPBMC (1x10\n6 cells/mL) in CO 2 (5 %) atmosphere at\n37°C. The cells were cultured on a culture medium\ncomposed as follows: RPMI 1640 with addition of 10%\nv/v inactivated foetal calf serum (FCS, 56°C, 30 min),\n100 U/mL penicillin and 100 μg/mL streptomycin, or\nwith addition of phytohaemagglutinin – PHA (5 μg/mL).\nIL-10 concentration was analyzed in enzyme-linked\nJarosław Górski, Krzysztof Szyłło, Małgorzata Banasik, Przemysław Lewkowicz, Henryk Tchórzewski\n\nArch Med Sci 1, March / 2007 39\nTreg in women with endometriosis\nimmunosorbent assay – ELISA (R&D Systems, USA),\nusing the commercially available Endogen kit.\nSSttaattiissttiiccaall  aannaallyyssiiss\nFor all analyzed parameters within examined\ngroups mean value, standard deviation (SD) and\nstandard error of mean (SEM) were calculated (in\nIL-10 release level analysis). \nStatistical verification was performed by Student’s\nt-test, and Pearson’s correlation coefficient signifi-\ncance test. P≤0.05 was assumed to be statistically\nsignificant. Statistical analysis was performed using\nSTATISTICA 5.0 PL software (StatSoft, Poland).\nResults \nThe number of deliveries and the main disorders in\nthe groups have been analysed additionally. The most\nnumerous part of this group is nonpartum women:\n12 patients altogether (50%), 4 in the control group \n(33.33%). Together with the advance of endometriosis\nthe number of nonpartum women rises. In the group\nof women with I/II° of endometriosis were 4 nonpartum\nwomen (33.33%) and in the group with III/IV° there\nwere 8 (66.66%). Irrespective of the degree of\nendometriosis, among women who gave birth, the\nmost common were those who gave birth once -\n10 women (41.66%). In comparison with the control\ngroup there are noticeable differences in the figure of\nsequent pregnancies: multipara from the examined\ngroup - 8.33%; in the control group - 25%. \nThe main disorder because of which a women\nreported to a gynaecologist was pelvic pain - it\noccured in 17 patients (70.83%). It is worth noting\nthat it is an atypical disorder as in the control group\nthe problem was reported by 9 women (75%).\nDifficulties with fertility were another problem -\n11 women (45.83%) were treated for infertility. \n4 women (16.66%) reported dyspareunia and 5 women\n(20.83%) reported dysmenorrhoea. The two last\nmentioned disorders coexisted with pelvic pain or\ninfertility. The detailed clinical profiles of the examined\ngroups are shown in Table I.\nIn the analysis of percentages of each T\nlymphocyte population in peripheral blood of\npatients with endometriosis and healthy subjects \na decrease in percentage of CD8\n+ lymphocytes in\naffected women was observed (Table II). Because\nTTaabbllee  II..  The clinical data of the examined groups\nPPaarraammeetteerr  eexxaammiinneedd  CCoonnttrrooll  ggrroouupp EEnnddoommeettrriioossiiss\nAAGGEE\nrange /years/ 17-47 22-52\naverage /years/ 36 34\nPPLLAACCEE  OOFF  RREESSIIDDEENNCCEE\nurban areas  /%/ 83.4 91.7\nrural areas /%/ 16.6 8.3\nNNUUMMBBEERR  OOFF  DDEELLIIVVEERRIIEESS\n0# n 4 12\n%3 3 . 3 3 5 0\n1# n 5 10\n% 41.66 41.66\n≥2# n3 2\n%2 5 8 . 3 3\nDDIISSOORRDDEERRSS\npelvic pain\nn 9 75\n% 17 70.83\ninfertility\nn 2 16.66\n% 11 45.83\ndyspareunia\nn 1 4\n%8 . 3 3 1 6 . 6 6\ndysmenorrhoea  \nn 2 5\n% 16.6 20.83\nn – number of patients\n# – number of deliveries\nTTaabbllee  IIII..  The distributions of T lymphocytes CD8+, CD4+, CD4+CD25+ in peripheral blood and peritoneal fluid of women\nwith endometriosis and healthy subjects (control group). Data are presented as means ± standard deviation (SD)\nPPaarraammeetteerr  eexxaammiinneedd  CCoonnttrrooll  ggrroouupp EEnnddoommeettrriioossiiss EEnnddoommeettrriioossiiss ((II--IIII°°)) EEnnddoommeettrriioossiiss ((IIIIII--IIVV°°))\n((nn==1122)) ((nn==2244)) ((nn==1122)) ((nn==1122))\nPPeerriipphheerraall  bblloooodd\nCCDD88++ ((%%)) 34.2 ± 5.71 28.0 ± 5.74 # 25.9 ± 7.20# 27.8 ± 7.50#\nCCDD44++ ((%%)) 45.8 ± 7.30 45.1 ± 8.19 46.2 ± 6.98 45.2 ± 7.97\nCCDD44//CCDD88--iinnddeexx 1.38 ± 0.35 1.78 ± 0.51 # 1.91 ± 0.54# 1.87 ± 0.82#\nCCDD44++CCDD2255++ ((%%)) 7.0 ± 3.06 8.3 ± 3.58 7.8 ± 2.30 8.7 ± 4.51*\nPPeerriittoonneeaall  fflluuiidd  \nCCDD88++ ((%%)) 50.1 ± 11.00 50.4 ± 13.20 50.2 ± 17.80 50.0 ± 5.74\nCCDD44++ ((%%)) 22.2 ± 9.40 18.8 ± 7.80 17.8 ± 7.30 # 20.0 ± 8.51\nCCDD44//CCDD88--iinnddeexx 0.40 ± 0.170 0.38 ± 0.150 0.36 ± 0.130 0.40 ± 0.180\nCCDD44++CCDD2255++ ((%%))  6.5 ± 2.89 6.3 ± 2.42 5.9 ± 1.83 6.67 ± 2.39\n# - p<0.05 statistically significant differences as compared to control group\n* - p=0.06 statistically significant differences as compared to control group\nn - number of performed investigations\n\n40 Arch Med Sci 1, March / 2007\nthe percentage of CD4+ lymphocytes was similar in\nall examined groups, the CD4/CD8 ratio turned out\nto be higher in affected subjects. Although\nCD4\n+CD25+ regulatory lymphocyte percentage in the\nperipheral blood did not differ significantly between\neach group, we still noted a higher increase in patients\nwith endometriosis (p=0.06). CD4\n+CD25+ lymphocyte\npercentage in peripheral blood rose together with\nadvancing stage of the disease (Figure 1). The analyses\ndid not indicate any shift in proportion of this\nsubpopulation examined in peritoneal fluid in\npatients with endometriosis in comparison to\nhealthy subjects. The percentage of these cells in\nperitoneal fluid also did not correlate with disease\nprogression (Figure 2).\nIL-10 generation by peripheral blood mononuclear\ncells was evaluated after the cells were cultured,\nwithout and after stimulation with PHA. Significantly\nlower production of this interleukin was observed in\nwomen suffering from endometriosis versus healthy\nsubjects (Figure 3). In PHA-stimulated cultures,\nsignificantly lower IL-10 production was observed\nonly in women with mild endometriosis. \nDiscussion\nIn the pathogenesis of endometriosis some natural\nand acquired humoral and cell-mediated immunity\ndisorders have been observed. Humoral response\ndisorders include the synthesis of abnormal antibodies\nto endometrial and ovary cells, phospholipids and\nhistones. Decreased cytotoxicity of NK cells,\nquantitative disorders between CD4 and CD8 cells, as\nwell as abnormal transformation of B lymphocytes\nare among the cellular response disorders [10].\nMany authors points to a local inflammatory\nreaction taking part in development of endometriosis,\nwith a special role of peritoneal fluid macrophages.\nCytokines and growth factors produced by these cells\naffect other morphotic elements, thus favouring ecto-\npic implantation, proliferation and angiogenesis [2, 3].\nSzyłło et al. [11] investigated the role of T\nlymphocytes in the pathogenesis of endometriosis.\nThey observed altered T cell redistribution between\nperipheral blood, peritoneal fluid and endometrial\ntissues. T lymphocytes in women with endometriosis\nproduce higher amounts of pro-inflammatory\ncytokines – mainly IL-6 and TNF-α, which can impair\nTreg cell function, thus causing the lack of defence\nagainst autoimmunization.\nThe isolation of CD4\n+CD25+ regulatory cells by\nSakaguchi et al. in 1995 brought the beginning of\nextensive studies on structure and use of these cells.\nThe authors claimed that the Treg cell population is\nresponsible for suppression of autoimmunization [4].\nCD4\n+CD25+ cells make up 5-10% of peripheral\nCD4+ T cells. The structure and operation of Treg cells\nhave been precisely described in many papers [5, 6,\n7, 8, 9]. Strength and mode of stimulation affect the\nfunctioning of regulatory cells [5, 6, 8, 12]. Present\nstudies focus on the role of these cells in individual\ndiseases, though there is a lack of manuscripts\nJarosław Górski, Krzysztof Szyłło, Małgorzata Banasik, Przemysław Lewkowicz, Henryk Tchórzewski\n0,5      1,0      1,5      2,0      2,5      3,0      3,5      4,0      4,5\nregresion 95% confidence interval\nbblloooodd\nssttaaggee\nFFiigguurree  11..  Analysis of Pearson’s linear correlation ratio\nbetween progression stage of the disease and percen-\ntage of Treg lymphocytes (CD4+CD25+) in peripheral\nblood\n20\n18\n16\n14\n12\n10\n8\n6\n4\n2\n0,5      1,0      1,5      2,0      2,5      3,0      3,5      4,0      4,5\nregresion 95% confidence interval\nbblloooodd\nssttaaggee\nFFiigguurree  22..  Analysis of Pearson’s linear correlation ratio\nbetween progression stage of the disease and percen-\ntage of Treg lymphocytes (CD4+CD25+) in peritoneal\nfluid\n14\n12\n10\n8\n6\n4\n2\nPBMC PBMC after \nPHA stimulation\ncontrol group\nendometriosis\nendometriosis (I–II)\nendometriosis (III–IV)\n*\n*\n[[ppgg//mmll]]\nFFiigguurree  33..  Analysis of IL-10 generation by peripheral\nblood mononuclear cells (PBMC) in patients with endo-\nmetriosis in comparison with healthy women\n1000\n500\n0\n**\n113\n66 46 61\n512 483\n400\n520\n\nArch Med Sci 1, March / 2007 41\ndescribing the role of Treg cells in endometriosis. The\nperitoneal fluid milieu, specific and having a major\nrole in development of endometriosis, was not\ninvestigated with respect to its effect on regulatory\nfunctions of the immune system.\nBased on the results we can observe a shift of\nCD4\n+CD25+ cells in peripheral blood of affected\npatients in comparison with healthy subjects. The\npercentage of Treg cells in peripheral blood increases\ncompared to healthy women. The values are close\nto the borderline of statistical significance (p=0.06).\nSuch a quantitative shift is not observed in the\nperitoneal fluid between the groups.\nIt was shown that Treg cells take part in\nconstraining the inflammation [5, 6, 8]. CD4\n+CD25+\ncells are responsible for effector cell (CD8 +)\nregulation. CD8 + cells, which are inhibitory in\nfunction, are suppressed by CD4+CD25+ cells. \nChanges in CD4 +CD25+ numbers influence CD8+\nshift. In the peritoneal fluid of women with\nendometriosis the percentage of Treg cells is lower\nthan in the peripheral blood, which might explain\nwhy the number of CD8\n+ cells in the peritoneal fluid\nis increased in comparison with the peripheral blood.\nAn unknown defect of CD4 +CD25+ cells or their\nimpaired migration in the inflammation site (which\nmay be a result of abnormal functioning of chemo-\nkines, for example) could lead to the lack of their \ninflammation-limiting action.\nDue to the lack of reports describing Treg cell\ninvolvement in endometriosis it is not possible to\ncompare our results with other studies. There is some\nresearch on the role of CD4\n+CD25+ Treg cells in other\ndiseases: among them we should mention atopic\ndermatitis [13], rheumatoid arthritis [14], juvenile\nidiopathic arthritis [15], type-1 diabetes [16, 17],\nmultiple sclerosis [18] and myasthenia gravis [19].\nIn patients with recurrent aphthous ulcerations\n(RAU) an increase in IL-6 and TNF-α pro-inflamma-\ntory cytokine levels and a decrease in IL-10 and TGF-β\nanti-inflammatory cytokine levels was observed [20].\nThere was a decrease in number of Treg suppressive\ncells in affected patients in comparison with healthy\nsubjects. It favoured inflammation as a response to\nbacterial antigens of microorganisms colonizing the\noral cavity. It is conceivable that a similar situation\noccurs in the case of endometriosis, where impaired\nfunction of Treg cells promotes pro-inflammatory\ncytokines, growth factors and antigens, which leads\nto survival, proliferation and development of\nendometrial tissue in the altered environment. \nThe number of CD4\n+CD25+ cells in the population\nof patients with myasthenia gravis [19] and multiple\nsclerosis [18] does not differ from the control group.\nSuppressive activity of CD4\n+CD25+ regulatory cells is\nweakened, with accompanied lowered FOXP3\nexpression [13]. Also, the secreted cytokines profile\nis changed. Some authors claim that high\nconcentration of IL-6 leads to loss of suppressive\nproperties [21].\nIn patients with atopic dermatitis it was observed\n[13] that CD4\n+CD25+ Treg expression is increased in\nperipheral blood in comparison with the control group,\nwith an accompanying rise in FOXP3 expression.\nBacterial antigens in the skin led to suppressive activity\nof Treg being reverted and their function impaired.\nDepending on the disease, the percentage of Treg\ncells in peripheral blood can rise [20], show no\nchange [13, 14, 17, 18] or fall [15, 16] in comparison\nwith healthy subjects.\nMany authors describe increased IL-10 level in\nperitoneal fluid of women with endometriosis in\ncomparison with the control group [22, 23, 24]. They\nrightly conclude that the main source of IL-10 is\nperitoneal macrophages. Our studies confirm this\nassumption, as the lowered number of CD4\n+ and\nCD4+CD25+ cells in the peritoneal fluid cannot make\nup for the main source of the cytokine production. \nIn the present study we observed a decreased\namount of IL-10 in peripheral blood in women with\nendometriosis. The increase after PHA stimulation\nwas lower in women suffering from endometriosis\nthan in the control group. Apart from anti-inflammatory\nproperties, IL-10 also takes part in suppression. While\nanalyzing Treg action using IL-10 one can conclude\nthat lowered IL-10 level in the peripheral blood results\nfrom impaired functioning of CD4\n+CD25+ Treg cells in\nthis disease, because in the peripheral blood of women\nwith endometriosis the percentage of CD4\n+CD25+ cells\nincreases. In current studies the percentage of Treg\ncells in peripheral blood increases independently of\nendometriosis progression, without an accompanying\nIL-10 rise in the peripheral blood, as was observed in\nprevious studies [22, 23, 24]. Most probably, in this\nspecific disease there are involved some mechanisms\nupsetting the activation and functioning of CD4\n+CD25+\ncells in peripheral blood, which can lead to lower levels\nof IL-10. Its decreased amount in peripheral blood\npromotes inflammation via lack of suppressive action.\nBecause of the small size of the investigated\ngroups, further observations are necessary in order\nto confirm the obtained results.\nConclusions\nWe analyzed the percentage of CD4+CD25+ T cells\nin peripheral blood and peritoneal fluid of women\nwith endometriosis, without the evaluation of\nactivity, which should be determined in further\nstudies. In the present study we observed a higher\nproportion of CD4\n+CD25+ Treg cells in the peripheral\nblood of affected women in comparison with healthy\nsubjects. We did not observe such a shift in the\nperitoneal fluid between the groups. The quantitative\ndisproportion of CD4\n+CD25+ T cells between peripheral\nblood and peritoneal fluid can affect the onset and\ndevelopment of endometriosis. The impairedmigration\nTreg in women with endometriosis\n\nand function of Treg cells alters the composition of\nthe peritoneal fluid, which favors the onset and\ndevelopment of endometriosis. Activity and maturity,\nconfirmed by increased expression of HLA-DR,\nCTLA-4, GITR markers, as well as FOXP3 expression,\nrequire further research.\nReferences\n1. Child TJ, Tan SL. Endometriosis aetiology, pathogenesis and\ntreatment. Drugs 2001; 61: 1735-1750.\n2. Oral E, Arici A. Pathogenesis of endometriosis. Obstet\nGynecol Clin North Am 1997; 24: 219-33.\n3. 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