{"paper_id":"0872d804-08d4-447e-8d74-3abe3f42a783","body_text":"R E S E A R C H A R T I C L E Open Access\nExogenous activated NK cells enhance\ntrafficking of endogenous NK cells to\nendometriotic lesions\nMary Lourdes Montenegro 1*, Rui Alberto Ferriani 1 and Per H. Basse 2\nAbstract\nBackground: Endometriosis is defined as the presence of endometrial glands and stroma at ectopic locations.\nAlthough the prevalence of endometriosis is as high as 35 % –50 %, its pathogenesis remains controversial.\nAn increasing number of studies suggest that changes in immune reactivity may be primarily involved in the\ndevelopment of endometriosis development. In this sense, it has been strongly suggested that a fundamental part of\nimmunologic system, the natural killer cells (NK cells), are an important part of this process. NK cells, a component of\nthe innate immune system, have been extensively studied for their ability to defend the organism against infections\nand malignancy. Recent studies have shown that IL-2-activated NK (A-NK) cells are able to attack and destroy tumors in\nlungs and livers of mice, demonstrating the therapeutic potential of these cells. Similarly to metastatic tumor cells,\nendometrial cells are able to adhere, infiltrate and proliferate at ectopic locations. Therefore, in this study, we evaluated\nthe ability of adoptively transferred and endogenous NK cells to infiltrate endometriosis lesions.\nMethods: As NK cells donors were used C57BL/6 B6. PL- Thy 1.1 female mice. As uterine horns donors were used C57/BL6\n+GFP female mice and as endometriosis recipients C57BL/6 Thy1.2 female mice. Endometriosis induction was made by\ninjection of endometrial tissue fragments. After 4 weeks, necessary for endometriosis lesions establishment the animals were\ndivided in 3 experimental groups with 10 animals each. Group 1 received i.v doses of 5x106 A-NK in 200μlR P M I ;G r o u p2\nreceived i.p dose of 5x106 A-NK in 200μl RPMI and Group 3 received i.p dose of IL2 (0.5 mL RPMI containing 5.000U of IL2).\nResults:Our data show that exogenous A-NK cells injected via ip combined with endogenous A-NK cells seems to be the\nmost efficient way for activated NK cells track and infiltrate endometriosis.\nConclusion:For the first time, it was shown that both endogenous as exogenous A-NK cells are able to track, migrate and\ninfiltrate endometriosis lesion. This seems to be a promising result, and if confirmed the efficiency of A-NK cells in killing\nendometriosis lesions, maybe in the future we could use this approach as an alternative treatment for women with\nendometriosis.\nKeywords:Endometriosis, NK cells, Activated NK cells, A-NK cells, Treatment\nBackground\nEndometriosis is cracterized by presence of endometrial\nglands and stroma in ectopic locations, as pelvic periton-\neum, ovaries, and rectovaginal septum, affecting 6 % to\n10 % of women in reproductive age [1]. Endometriosis\ncan cause dysmenorrhea, dyspareunia, chronic pelvic\npain and infertility [1]. The prevalence of endometriosis\nin women experiencing pain, infertility, or both is as\nhigh as 35 % –50 % [2]. The pathogenesis of endometri-\nosis remains controversial. Currently, an increasing\nnumber of studies have addressed whether changes in\nimmune reactivity may facilitate development of endo-\nmetriosis [3 –5]. In this sense, it has been strongly sug-\ngested that a fundamental part of immunologic system,\nthe natural killer cells (NK cells), are an important part\nof this process [6, 7].\nNK cells are large granular lymphocytes, representing\nabout 5 % to 15 % of peripheral blood lymphocytes [8].\n* Correspondence: montenegromlls@gmail.com\n1Department of Gynecology and Obstetrics of Faculty of Medicine of\nRibeirão Preto, University of São Paulo, Ribeirao Preto, SP, Brazil\nFull list of author information is available at the end of the article\n© 2015 Montenegro et al. This is an Open Access article distributed under the terms of the Creative Commons Attribution\nLicense (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any\nmedium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://\ncreativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.\nMontenegro et al. BMC Immunology  (2015) 16:51 \nDOI 10.1186/s12865-015-0105-0\n\nFig. 1 Experimental endometriosis lesions 4 weeks after injection of endometrial tissue. Donor mice: GFP transgenic C57BL/6; Recipient mice:\nwild-type (wt) C57BL/6. All pictures show endometriosis lesions from different animals\nFig. 2 GFP-positive endometriosis lesions. Experimental endometriosis lesions were induced by i.p injection of fragments of endometrium from GFP\ntransgenic C57BL/6 mice. The lesions were removed 4 weeks later, fixed in 4 % paraformaldehyde, frozen and sectioned. Sections were stained with\nHoechst 33342 to reveal cell nuclei. The endometriosis tissue is strongly positive for GFP, proving that the tissue is of donor origin. Image resolution-\n20X. All micrographs show endometriosis lesions from different animals\nMontenegro et al. BMC Immunology  (2015) 16:51 Page 2 of 7\n\nNK cells are an important component of the innate\nimmune system and have been extensively studied for\ntheir ability to defend the organism against infections\nand malignancies [9]. NK cells kill their targets by dir-\nect lysis or by the release of cytokines and chemokines\n[10]. These reactions are carefully balanced and the\nparticipation of cytokin es such as interferons α and β\nand interleukin 2 (IL-2) are essential [11]. In addition\nto increasing the traffic of NK cells to sites of injury,\nIL-2 activation has a strong proliferative effect on NK\ncells [3]. Studies have shown that NK cells activated by\nIL-2 (Adherent NK cells or A-NK cells) are able to\ninfiltrate and destroy tumors in lungs and livers of mice\n[10, 11], suggesting that activated NK cells may be of\ntherapeutic importance in the setting of cancer. Recently,\na study demonstrated that activation of leucocytes with\nIL-2, induced a persistent reduction of endometriosis\nlesions in female rats [12]. On this basis, is possible that\nIL-2 activated NK cells could form the basis of a new\ntreatment alternative for patients with endometriosis.\nResults\nOur analyses show that exogenous A-NK cells injected\nvia the i.v or i.p routes very efficiently infiltrate endo-\nmetriosis lesions (Figs. 3, 4). In non-treated animals,\nendogenous NK cells are only found in very low\nnumbers in endometriosis lesions (Fig. 5). However, in\nanimals treated with A-NK cells plus IL-2 or with IL-2\nalone, the density of endogenous NK cells in the endo-\nmetriotic lesions increased at least 10 fold (Figs. 6, 7).\nDiscussion\nEndometriosis affects almost 10 % of women in the repro-\nductive age. The pathogenesis of endometriosis remains\ncontroversial. However, many recent studies have impli-\ncated the immune system and especially NK cells in\nendometriosis development. On this basis, NK cells, a\nfundamental part of immunologic system seems to play an\nimportant role in endometriosis development. Several of\nthese studies have reported a decrease in cytotoxicity of\nNK cells in the peritoneal fluid from women with endo-\nmetriosis [12, 13, 18]. Lack of NK cell cytotoxicity could, at\nleast in part, facilitate the attachment of endometriotic cells\nat ectopic sites [6]. However, the reason for the decrease in\ncytotoxicity of peritoneal fluid NK cells remains to be\nclarified, but only few studies have assessed the role of NK\ncells in target endometriosis lesions. Velasco and Cols\nfound that treatment of female rats with endometriosis\nwith IL-2 (two doses given by the i.p. route) resulted in the\nactivation of leucocytes and a significant reduction in the\nsize of endometriotic lesions compared to the untreated\ngroup [12]. However, when they look for activated NK\nFig. 3 Exogenous A-NK cells injected intravenously localize at sites of endometriosis lesions. Fresh frozen endometriosis tissue was sectioned and\nstained with PE anti-CD90.1 to reveal the Thy1.1+, adoptively transferred A-NK cells in the Thy1.2+ recipients. Sections were stained with Hoechst\n33342 to reveal cell nuclei. Image resolution 20X. All micrographs ( a-d) show endometriosis lesions from different animals\nMontenegro et al. BMC Immunology  (2015) 16:51 Page 3 of 7\n\ncells, it seems to be in low number compared with control.\nDifferently, in our study we show that when activated by\nIL2, endogenous NK cells starts to migrate to the lesion\nbecoming able to infiltrate it and that this effect seem to be\npotentiate when exogenous A-NK were injected via i.v or\ni . p .T h i si m p o r t a n te v i d e n c es h o w na tl e a s ti np a r tt h a tN K\ncells seem really play an important role on pathogenesis of\nendometriosis. We believe that ours findings were different\nfrom those found for Velasco ’s, first due the difference in\nexperimental model, and second due the difference in pro-\ntocols accessed by each study. While we collected tissue\nthree days after A-NK cells induction, the other study wait\n3m o n t h st oc o l l e c tt h es i m p l e s ,m a y b et h i sl o n gt i m eh a s\ninfluenced in activity and quantity of NK cells. On the\nother hand, due our short-term protocol, was not possible\nobserve changes in lesions size. Despite of this, for the first\ntime we show that activated NK cells are able to track and\ninfiltrate endometriosis lesions and the best way to pro-\nmote this effect is through combination of endogenous\nA-NK cells and exogenous A-NK cells injected via i.p.\nConclusions\nOur results show at least in part that exogenous activated\nNK cells enhance trafficking of endogenous NK cells to\nendometriotic lesions. It seems to be a very promising\nresult, and if confirmed that A-NK cells are efficient in\nkilling endometriosis lesions, maybe in the future we\ncould use this approach as an alternative treatment for\nwomen with endometriosis. Therefore, we believe it is ne-\ncessary understand better decreasing of cytotoxicity of NK\ncells in peritoneal fluid of women with endometriosis and\nwhy endogenous NK cells seems not to be able to attack\nendometriosis and also if exogenous A-NK cells despite\ninfiltrate the lesions, are really able to kill endometriosis.\nMethods\nThe study was performed at University of Pittsburgh\nCancer Institute and approved by University ethics com-\nmittee under the Protocol N o 12091050.\nSplenocytes from C57BL/6 B6. PL- Thy 1.1 female mice\nwere used for production of A-NK cells. As uterine horn\ndonors we used C57/BL6 + GFP female mice (transgenic\nfor green fluorescent protein) (Fig. 2) and endometriosis\nwas established in C57BL/6 (Thy1.2) female mice. All\nmice were 8-12 weeks of age.\nEndometriosis was induced by i.p. injection of endo-\nmetrial tissue fragments, according Wieser et al [13].\nTo stimulate proliferation of endometrial tissue, GFP\ndonor mice received estradiol valerate subcutaneously\n(100 μg/kg dissolved in corn oil) one week before harvest\nof uterine tissue. Following harvest, uterine horns were\nopened and placed in a sterile Petri dish with PBS [15].\nFig. 4 Exogenous A-NK cells injected intraperitoneally localize at sites of endometriosis lesions. Fresh frozen endometriosis tissue was sectioned\nand stained with PE anti-CD90.1 to reveal the i.p. injected, Thy1.1+ A-NK cells in the Thy1.2+ recipients. Sections were stained with Hoechst 33342\nto reveal cell nuclei. Image resolution 20X. All micrographs ( a-d) show endometriosis lesions from different animals\nMontenegro et al. BMC Immunology  (2015) 16:51 Page 4 of 7\n\nThe endometrium was detached from the uterine muscle\nand finely chopped with a scalpel [16, 17]. Finally, the\nendometrial fragments were suspended in 0.6 ml PBS and\ninjected i.p into recipient mice using an 18 gauge dispos-\nable needle [13]. Each recipient mouse received an equal\nquantity of tissue (~40 mg). Recipient mice also received\n100 μg / k ge s t r a d i o lv a l e r a t es u b c u t a n e o u s l yo n c eaw e e k ,\nstarting one week before receiving the endometrial tissue.\nThis procedure was performed in order to synchronize\ntheir estrous cycles. After 4 weeks, GFP-positive endomet-\nriosis lesions were established (Fig. 1) and the experimen-\ntal protocol was started [18].\nFig. 5 IL-2 treatment augments infiltration of endometriosis lesions by endogenous NK cells. Fresh frozen endometriosis tissue from animals\nreceiving Peg-IL-2 only was sectioned and stained with anti-NKp46 (or isotype control) followed by Alexa 488-conjugated donkey anti-goat\nantibody to identify endogenous NK cells Sections were also stained with Hoechst 33342 to reveal cell nuclei. Image resolution 20X. ( a-b) Isotype\ncontrol plus Alexa 488 donkey anti-goat. ( c-d) Staining with anti-NKp46 plus Alexa 488 donkey anti-goat antibody reveals a substantial infiltration\nof the lesions by endogenous NK cells. Image resolution 20X\nFig. 6 Infiltration of endometriosis lesions by endogenous NK and exogenous A-NK cells. Sections from endometriosis lesions were double-\nstained with PE anti-Thy1.1 antibody and anti-NKp46 antibody followed by Alexa 488-conjugated donkey anti-goat antibody to identify adoptively\ntransferred A-NK cells (Thy1.1+/ NKp46+) and endogenous NK cells (Thy1.1-/NKp46+). Slides were stained with Hoechst 33342 (for nuclei). Image\nresolution 20X. ( a) Endometriosis lesion from animal that received exogenous A-NK cells i.p. ( b) Endometriosis lesion from animal that received\nexogenous A-NK cells by the i.v. route\nMontenegro et al. BMC Immunology  (2015) 16:51 Page 5 of 7\n\nExperimental protocols\nAnimals were divided in 3 experimental groups with 10\nanimals each. Group 1 received i.v doses of 5 × 10 6 A-NK\nin 200 μl RPMI; Group 2 received i.p dose of 5 × 10 6 A-\nNK in 200 μl RPMI; Group 3 received i.p dose of IL2\n(0.5 mL RPMI containing 30.000 IU/mL of pegylated\n(PEG-) rhIL2). To support th e transferred A-NK cells,\nanimals from group 1 and 2 received i.p. injections of\n0.5 mL RPMI containing 30.000 IU/mL of pegylated\n( P E G - )r h I L 2a t1 2h o u ri n t e r v a l sf o r3d a y s .G r o u p3\nreceived the same amount of Peg-IL-2 (IL-2 control),\nwhereas control group received injections of 0.5 ml\nRPMI without IL-2 [14].\nA-NK cells preparation\nSpleens were removed aseptically from C57BL/6 B6. PL-\nThy 1.1 female mice and a single-cell suspension was\nprepared in RPMI1640. Erythrocytes were lysed by incu-\nbation with ammonium chloride-potassium buffer at\nroom temperature for 3 min and the spleen cells were\nsubsequently washed twice in RPMI1640. Cells were\ntransferred to T150 plastic flasks (Falcon, B&D, Franklin\nLakes, NJ, USA) and cultured at 37 °C in an atmosphere\nof 5 % CO 2 in 50 ml of RPMI1640 supplemented with\n5 % heat inactivated fetal calf serum and 5 % normal hu-\nman serum, 10 ml/l non-essential amino acids (Life\nTechnologies), 50 mM 2-mercaptoethanol, 2 mM glu-\ntamine, 20 mM Hepes buffer, 0.8 g/l streptomycin and\n1.6x105u/l penicillin, hereafter referred to as complete\nmedium (CM). Cells were stimulated with 6.000U/mL of\nhuman recombinant IL-2. After 3 days of incubation,\nCD8-positive cells were magnetically removed following\nincubation of the cell culture with rat anti-CD8 antibody\n(ATCC, TIB-105) and subsequently with anti-rat coated\nmagnetic beads (Dynal Biotech, Lake Success, NY, USA).\nThe CD8-depleted cells were resuspended in fresh CM\ncontaining 6000 IU/mL IL-2 to a final concentration of\n1x105cells/ml and returned to culture flasks. After an\nadditional 3 days of culture, non-adherent cells were\ndecanted and the plastic-adherent cells were harvested\nafter a brief treatment with 0.02 % EDTA and washed\ntwice in RPMI1640 before use [11]. Routinely, these A-\nNK cells were 95 % Thy1.1, 95 % asGM1, 90 % NK1.1,\n2 % CD8 and 2 % CD4 [11, 14].\nAdoptive transference of A-NK cells\nA-NK cells were adjusted to appropriate concentrations\nand injected into C57BL/6 Thy1.2 mice via lateral tail vein\n(i.v.) or into the peritoneal cavity (i.p.). To support the\nt r a n s f e r r e dA - N Kc e l l s ,a n i m a l sf r o mg r o u p1a n d2\nreceived i.p. injections of 0.5 mL RPMI containing\n30.000 IU/mL of pegylated (PEG - )r h I L 2a t1 2h o u ri n t e r -\nvals for 3 days. Group 3 received the same amount of\nPeg-IL-2 (IL-2 control), whereas group 4 received injections\nof 0.5 ml RPMI without IL-2.\nAccumulation of exogenous and endogenous NK cells in\nendometriosis lesions\nAt three days after injection of A-NK cells, animals were\nsacrificed and the lesions rem oved. The collected tissues\nwere sectioned and fixed in ice-cold acetone for 5 minutes.\nSections were stained with PE anti-rat CD90.1 to reveal the\nThy1.1+, adoptively transferred A-NK cells in the Thy1.2+\nrecipients. Sections we also stained with anti-NKp46 anti-\nbody followed by Alexa 488 donkey anti-goat antibody to\nreveal endogenous NK cells (NKp46+/Thy1.1-). Before\nFig. 7 Density of endogenous and exogenous NK cells at sites of endometriosis. Endometriosis tissue was cut and stained as described in the\nlegend to Fig. 6. ( a) Endometriosis lesion from control animal (no treatment) ( b) Endometriosis lesion from animal that received PEG-IL2 only. ( c)\nEndometriosis lesion from animal that received i.v. A-NK cells by the i.v. route. ( d) Endometriosis lesion from animals that received i.p. injection of\nA-NK cells. Image resolution 20X. Slides were stained with Hoechst 33342 (for nuclei). ( e) Exogenous and endogenous NK cells were identified as\nPE-positive and Alexa 488-positive, PE-negative cells, respectively, and counted. Results are presented as mean number of cells/field of vision ±\nSD. Endogenous NK cells (black columns), exogenous A-NK cells (gray columns)\nMontenegro et al. BMC Immunology  (2015) 16:51 Page 6 of 7\n\ncoverslipping, sections we stained with Hoechst 33342 to\nreveal all nuclei.\nThe density of immune-stained cells were determined\nby image analysis (MetaMorph ®) [14].\nStatistical analysis\nTo compare all study groups, analysis of variance (ANOVA)\nwas performed.\nAbbreviations\nNK cells: Natural killer cells; IL2: Interleukin 2; i.v: Intravenous;\ni.p: Intraperitoneal; PEG-IL2: Human recombinant IL2 complexed to\npolyethylene glycol; GFP: Green Fluorescent Protein; A-NK cells: Activated\nNatural Killer Cells.\nCompeting interests\nThe authors declare that they have no competing interests.\nAuthors’ contributions\nMLM, PHB and RAF conceived the idea; MLM and PHB did the literature\nsurvey; MLM run the experiments and writes the manuscript; all authors\nanalyze the data, read and approve the final manuscript.\nAcknowledgments\nThe authors wish to thank MSc Lisa Bailey for her valorous help during the\nexperiments and to Fundação de Amparo a Pesquisa do Estado de São\nPaulo- FAPESP for funding the research.\nThis project used the UPCI Cell and Tissue Imaging Facility (CTIF), a shared\nresource that is supported in part by award P30CA047904 from the NIH.\nAuthor details\n1Department of Gynecology and Obstetrics of Faculty of Medicine of\nRibeirão Preto, University of São Paulo, Ribeirao Preto, SP, Brazil. 2Department\nof Immunology of University of Pittsburgh, Pittsburgh, PA, USA.\nReceived: 20 January 2015 Accepted: 26 June 2015\nReferences\n1. 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Fertil Steril. 1997, 67(5):817 –21.\nSubmit your next manuscript to BioMed Central\nand take full advantage of: \n• Convenient online submission\n• Thorough peer review\n• No space constraints or color figure charges\n• Immediate publication on acceptance\n• Inclusion in PubMed, CAS, Scopus and Google Scholar\n• Research which is freely available for redistribution\nSubmit your manuscript at \nwww.biomedcentral.com/submit\nMontenegro et al. BMC Immunology  (2015) 16:51 Page 7 of 7","source_license":"CC0","license_restricted":false}