Flow Cytometric Evaluation of Intracellular Cytokine Synthesis in Peripheral Mononuclear Cells of...

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Immunological Investigations, 37:43–61, 2008Copyright © Informa Healthcare USA, Inc.ISSN: 0882-0139 print / 1532-4311 onlineDOI: 10.1080/08820130701554962

LIMM0882-01391532-4311Immunological Investigations, Vol. 37, No. 1, November 2008: pp. 1–28Immunological InvestigationsFlow Cytometric Evaluation of Intracellular Cytokine Synthesis in Peripheral Mononuclear Cells of Women with EndometriosisIncreased Expression of MCP-1 in women with endometriosisG. B. Gmyrek et al.

Grzegorz B. Gmyrek,1 Urszula Sieradzka,2 Marian Goluda,2 Marian Gabrys,2 Rafal Sozanski,3 Malgorzata Jerzak,4 Iwona Zbyryt,5 Agnieszka Chrobak,1 and Anna Chelmonska – Soyta1,5

1Laboratory of Reproductive Immunology, Institute of Immunology and ExperimentalTherapy, Polish Academy of Sciences, Wroclaw, Poland2Second Department of Obstetrics and Gynecology, Wroclaw Medical University,Wroclaw, Dyrekcyjna 5/7, Wroclaw, Poland3First Department of Obstetrics and Gynecology, Wroclaw Medical University,Wroclaw, Poland4Department of Gynecology, Military Institute of Medicine, Warsaw, Poland5Wroclaw University of Environmental and Life Sciences, Veterinary MedicineFaculty, Wroclaw, Poland

Systemic changes related to cytokine expression levels in women with endometriosisremain a subject of controversy. There are many studies concerning this topic showingdifferential serum cytokine levels; however, there are limited data presenting cytokineexpression at the single-cell level. This study focused on this question by measuringintracellular cytokine staining of activated peripheral CD3+ and CD14+ cells fromwomen with endometriosis (investigative group) compared with those with uterine lei-omyoma (control group). Isolated peripheral blood mononuclear cells from women withendometriosis and uterine leiomyoma were stimulated with PMA and ionomycin orwith LPS to induce intracellular synthesis of TNF-alpha, IFN-gamma, and IL-8 in sub-populations of CD3+ cells and TNF-alpha, IL-6, IL-10, MCP-1, and IL-8 in CD14+ cells.Comparison of the total groups of patients showed no significant differences in any ofthe intracellular cytokines investigated in the T cells and monocytes of women withendometriosis compared with controls. When the group of women with endometriosiswas divided with regard to severity of disease, a significantly lower percentage of

Address correspondence to Grzegorz B. Gmyrek, Institute of Immunology and Experi-mental Therapy, Laboratory of Reproductive Immunology, R. Weigla 12, 53-114,Wroclaw, Poland; E-mail: [email protected]

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44 G. B. Gmyrek et al.

CD3+CD8- lymphocytes stained for IFN-gamma and a significantly higher percentageof CD14+ cells stained for MCP-1 in advanced endometriosis patients compared withthe control group were observed. We conclude that peripheral mononuclear cells inwomen with advanced endometriosis may have differential cytokine synthesis in vitro.These results support the idea that differing immune cell activity measured byintracellular cytokine profiles in women with advanced endometriosis may be more aconsequence of the disease than a cause.

Keywords Endometriosis, Intracellular Cytokine staining, Flow cytometry, Uterineleiomyoma.

INTRODUCTION

The involvement of immunological aspects related to systemic changes incell-mediated immunity in the pathogenesis of endometriosis is still a subjectof controversy. These changes relate to alterations in T lymphocyte, monocyte,and NK cell function based on the following observations: (1) a decreasedactivity of peripheral NK cells, (2) an impaired cytotoxicity of peripheral NKand T cells to autologous endometrium (Berkkanoglu and Arici, 2003), (3) anincreased ability of peripheral blood monocytes of endometriosis patients toproduce proinflammatory cytokines both in basal and stimulated conditionscompared with healthy women (Dmowski et al., 2004), (4) a decreased chemo-tactic index of peripheral blood polymorphonuclear leukocytes (Garzetti et al.,1998), and (5) abnormalities in serum cytokine expression (Yang et al., 2004).All together, these observations could indicate a differential biological behav-ior of peripheral leukocytes in women with endometriosis in contrast to thosewithout this disease and provide a rationale for considering endometriosis asystemic disease. However, it is still unclear whether systemic immune cellactivity is more a cause of endometriosis or a consequence of the course of thedisease.

The subject of our study was the investigation of cytokines known to beimportant in the course of the endometriosis: tumor necrosis factor–alpha(TNF-alpha), gamma interferon (IFN-gamma), interleukin-8 (IL-8), mono-cyte chemotactic protein-1 (MCP-1), interleukin-6 (IL-6), and interleukin-10(IL-10) (Wu et al., 2003, Kayisli et al., 2002). In general, all of these cytok-ines are involved in the regulation of the inflammatory process andinterplay as a complex network (Kayisli et al., 2002; Whiteside, 1994). Theyhave been extensively investigated in women with endometriosis; however, acomparison of published reports reveals differences. Some show significantlyelevated MCP-1, TNF-alpha, IL-8, and IL-6 levels in serum (Pizzo et al.,2002; Gmyrek et al., 2005; Akoum et al., 1996; Markowska et al., 2004),while other results show no significant differences in the serum levels ofIL-8 (Barcz et al., 2002) or IL-6 (Velasco et al., 2005) between endometriosispatients and controls.

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Increased Expression of MCP-1 in Women with Endometriosis 45

In most of these cases the main investigational technique was ELISA. Itsprincipal drawback is its difficulty in indicating which cell population isresponsible for increased or decreased levels of cytokine produced (Pala et al.,2000). These cytokines can be produced by various mononuclear cells, and thecytokine production of a cell population may not correspond to the expressionat the single-cell level (Whiteside, 1994; Prussin et al., 1995). Therefore,important are not only the concentrations of these cytokines in biologicalfluids, but primarily differences in the production of the cytokine of interest atthe single-cell level and the consequences of this finding for the pathogenesisof endometriosis. There are methods of measuring cytokine production at thesingle-cell level (Carter et al., 1997). One of them is intracellular cytokinestaining using flow cytometry. The great advantage of this assay is the possi-bility of displaying the heterogeneity of cell populations by observing theco-expression of different cytokines in individual cells at the same time (Palaet al., 2000).

In this study we examined whether the percentages of T cells and mono-cytes stained for any investigative cytokine are different in women withendometriosis compared with those without this disease. For this reason weemployed intracellular cytokine staining in stimulated peripheral T cells andmonocytes derived from endometriosis patients and compared them withthose of women with uterine leiomyoma, serving as a control group.

MATERIALS AND METHODS

Characteristics of the PatientsThe subjects of this study were pre-menopausal women with regular

menses undergoing surgical visualization for any gynecological disorder atthe First and Second Departments of Obstetrics and Gynecology, WroclawMedical University. In 36 women, 23 to 53 years of age, visiting the Depart-ments of Obstetrics and Gynecology with symptoms of infertility or pain,the existence of endometriosis was documented based on laparoscopicexamination. The intervention was performed by different surgeons, butalways using the same technical procedure. The initial diagnosis ofendometriosis, evaluated by a clinician at the time of laparoscopy, wasconfirmed in all cases by histopathological examination. The biopsysamples were stained with hematoxylin and eosin and analyzed with a lightmicroscope in all cases by an experienced pathologist. The severity of thedisease was determined according to the revised American Society forReproductive Medicine (rASRM) classification (American Society for Repro-ductive Medicine, 1996). Among the endometriosis patients were womenwith mild (rASRM stages I-II, n = 13) and advanced (rASRM stages III-IV,n = 23) stages of disease.

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Twelve women, with suspected leiomyomata uteri, 27 to 51 years of age,affected by dysmenorrhea, hypermenorrhea, or lower abdominal pain, hadhysterectomies via either the abdominal or vaginal route and showed uterineleiomyoma (n = 12). The final diagnosis was confirmed in all cases byhistopathological examination by the same pathologist as in the case of theanalysis of endometriosis samples. Seven women showed intramural and twoother patients subserous uterine leiomyomas. One woman had concurrentintramural, subserous, and submucosal uterine leiomyomas, whereas 2 otherpatients had intramural and subserous uterine leiomyomas simultaneously.None of the women had endometrial hyperplasia, adenocarcinoma, orsystemic lupus erythematosus (SLE). No evidence of iron-deficiency anemia(IRA) due to existing hypermenorrhea in women with uterine leiomyomas wasnoted. There is evidence that the activity of systemic immune cells may bechanged in persons with IRA (Bergman et al., 2004). However, in all patientswith uterine leiomyomas in this study, evidence of IRA was excluded based onserum ferritin level (mean ± SE: 22.9 ± 4.7 ng/ml).

The group of women with uterine leiomyomas served as the control groupin this study. Current literature provides examples using patients withuterine leiomyoma as a control group (Kusume et al., 2005; Izumiya et al.,2003). It also seems that serum cytokines, steroid hormones, and growth fac-tors do not differ in women with leiomyoma compared with healthy women(Gmyrek et al., 2005; Dawood et al., 1994). Although some data show signifi-cant differences in serum IL-1-alpha level in women with uterine leiomyomas(Sikorski et al., 2001), no one confirmed this finding later. Therefore it isbelieved that any alternations in cytokine or growth factor levels associatedwith uterine leiomyoma are more likely in the uterine myometrium environ-ment than in the peripheral blood (Gmyrek et al., 2005). Obtaining samplesfrom healthy women attending a clinic for tubal ligation was impossible assuch surgical interventions are restricted by law in Poland. Collectingsamples from women with benign disorders other than uterine leiomyomas,for example ovarian cysts without evidence of endometriosis, failed in thisstudy due to an insufficient number of samples (n<4) for statistical analysis.For this reason, uterine leiomyoma patients served as the control group ofchoice in this study. Women with uterine leiomyomas with co-existingadenomyosis (n = 3) or with adenomyosis alone (n = 2) were excluded from thestudy.

Phase-cycle dating was performed by dating the endometrium accordingto the criteria of Noyes et al. (Noyes et al., 1975) (uterine leiomyoma patients)or by measuring serum progesterone level (endometriosis patients). Serumprogesterone and ferritin levels were evaluated by a solid-phase two-sitechemiluminescent immunometric assay with IMMULITE 1000 and 2000 ana-lyzers, respectively (Diagnostics Product Corporation, Los Angeles, CA, USA).The detection limit of the assay for progesterone and ferritin was 0.1 ng/ml

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and 1.5 ng/ml, respectively. Among all patients, 6 of the 12 women withuterine leiomyoma and 14 of the 36 endometriosis patients were in the prolif-erative phase of the menstrual cycle, whereas the others were in the secretoryphase. None of the women had received blood transfusion or hormonal and/oranti-inflammatory medications at least three months prior to material collec-tion. The study protocol was approved by the Local Ethics Committee of theWroclaw Medical University and informed consent was obtained from eachwoman prior to sample collection.

Origin of Reagents and Basic EquipmentHistopague 1077, ionomycin, L-glutamine, penicillin, streptomycin, LPS

(026:B6 serotype from E. coli), and trypan blue were purchased from Sigma-Aldrich Corporation (St. Louis, MO, USA). Sodium azide and formaldehydewere purchased from POCH (Gliwice, Poland). Phorbol 12-myristate-13-acetate (PMA) originated from Biomol GmbH (Hamburg, Germany). Fetal calfserum was manufactured by Invitrogen GmbH (Karlsruhe, Germany). Theantibodies anti-CD3-PerCP (clone: SK7), anti-CD8-FITC (clone: SK1), anti-CD4-FITC (clone: SK3), anti-CD69-PE (clone: FN50), anti-CD14-FITC (clone:M5E2), anti-CD16-FITC (clone: 3G8), anti-human TNF-alpha-PE (clone:MAb-11), anti-human IL-6-PE (clone: MQ2-6A3), anti-human IL-10-PE (clone:JES3-19F1), anti-human MCP-1-PE (clone: 5D3-F7), anti-human IL-8-PE(clone: G265-8), and anti-human IFN-gamma-PE (clone: 4S.B3) and isotypecontrols (Mouse IgG1-PE, Rat IgG2a-PE, and Mouse IgG2b-PE) werepurchased from BD Biosciences (San Jose, CA, USA).

The antibodies anti-human HLA-DR-PE (clone: TÜ36) and anti-humanHLA-DR-PE (clone: L243) originated from Invitrogen (Carlsbad, CA, USA)and BD Biosciences (San Jose, CA, USA), respectively. Reagents for fixationand permeabilization (Fix & Perm) and intracellular transport inhibition(GolgiPlug) originated from Invitrogen (Carlsbad, CA, USA) and BDBiosciences (San Jose, CA, USA) respectively. Siliconed glass tubes contain-ing heparin and siliconed sterile tubes without clotting activator were fromBD Vacutainer Systems (Plymouth, UK). Round-bottom polypropylenesterile tubes and eppendorff tubes were purchased from Medlab (Raszyn,Poland).

Sample CollectionVenous blood (5–10 ml) was aspirated from women before induction of

anesthesia into sterile siliconed glass tubes containing heparin. Simulta-neously, 3–5 ml of blood was withdrawn to siliconed sterile tubes to obtainserum. All patients were fasting before blood drawing. Venous blood wascollected between 8:00 a.m. and 1:00 p.m., always by the same medicalemployee. The serum was transferred to polypropylene tubes (Eppendorff

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type) and frozen at –20°C until the start of serum progesterone (endometriosispatients) or ferritin level (uterine leiomyoma) determination.

Cell PreparationPeripheral blood mononuclear cells (PBMCs) were isolated by layering

over Histopaque and centrifuged at 1183 x g for 20 minutes. The isolatedPBMCs were washed twice with PBS without Mg2+ and Ca2+ to remove resid-ual Histopaque solution, counted manually with a Bürker camera, and thenreconstituted to a final density at 1.0 x 106 cells/ml in RPMI-1640 medium(containing 2 mM L-glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin)supplemented with 10% heat-inactivated (56°C for 30–45 minutes) fetal calfserum. The viability of PBMCs, verified with the trypan blue exclusion test,was typically more than 95%.

Basic Immunophenotypic Flow CytometryPBMCs were transferred into plastic tubes in amounts of 40 μl each at

densities of 0.2-0.5 x 106 cells per tube. For each patient, antibodies to thefollowing combinations were measured by 3-color and 2-color flow cytometry:CD3+CD4+CD69+, CD3+CD8+CD69+, CD16+, and CD14+HLA-DR+. Beforeadding the antibodies, the PBMCs were washed once in Staining Buffer (SB;PBS without Ca2+ and Mg2+ ions supplemented with 5% fetal calf serum and0.09% sodium azide). After adding the antibodies the tubes were gentlyvortexed and left in the dark at room temperature for 15 minutes.

Next, to remove unbound antibodies the PBMCs were washed once in SB,fixed with 1% formaldehyde, and stored at 4°C until flow cytometry acquisitiontime (no longer than 48 hours). The cells were gated according to side-lightscattering properties and positively selected for CD3+, CD16+, and CD14+.Then, within the CD3+ gate (CD3-PerCP vs. side scatter) and the CD14+ gate(CD14-FITC vs. side scatter) the percentages of CD3+CD4+CD69+ andCD3+CD8+CD69+ or CD14+HLA-DR+ cells were determined, respectively.

Cell Culture ConditionsPBMCs reconstituted in RPMI-1640 medium with 10% FCS (mean

concentration ~106/ml) were divided into 2 sterile round-bottom polypropylenetubes and cultured at 37°C in 5% CO2. To study the intracellular cytokineproduction in CD3+ cells, the PBMCs in the first tube were stimulated withPMA and ionomycin at concentrations of 50 ng/ml and 1 μg/ml, respectively.The cells were cultured for 6 hr and the cytokine secretion was blocked byGolgiPlug (containing 99.9% dimethylsulfoxide and 0.1% brefeldin A) at a con-centration of 2 μl for every 1 ml of cell culture. In the second tube the PBMCsuspension was treated with LPS at a concentration of 1 μg/ml for intracellular

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cytokine production by CD14+ cells. These cells were cultured for 9 hr and, toinhibit cytokine secretion, GolgiPlug was also added at a concentration of 2 μlfor every 1 ml of cell culture for the last 4 hr of cell culture incubation.

Intracellular Cytokine StainingPBMCs were harvested and washed once in SB, suspended, and

distributed to polystyrene tubes in small volumes (40 μl per tube) at a densityof 0.5–1.0 x 106 cells per tube. The mixture of CD3-PerCP and CD8-FITCantibodies or CD14-FITC antibody was added to the cell suspension at theconcentrations suggested by the manufacturer and incubated for 15 minutesin the dark at room temperature. After that the cells were washed with 2 ml ofSB, then fixed (15 minutes in the dark at room temperature) and permeabi-lized (20 minutes in the dark at room temperature) with a commercially avail-able kit (Fix&Perm). Between each step (fixation and permeabilization) thecells were washed with 2 ml of SB.

During the permeabilization step, TNF-alpha-PE, IFN-gamma-PE, orIL-8-PE fluorochrome-conjugated cytokine antibodies were added to the tubescontaining cells stained with CD3-PerCP and CD8-FITC. Fluorochrome-conjugated cytokine antibodies TNF-alpha-PE, IL-6-PE, IL-10-PE, MCP-1-PE,or IL-8-PE were added to the tubes containing cells stained with CD14-FITC.Then the cells were incubated for 20 minutes in the dark at room temperatureand then washed once with SB to remove unbound cytokine antibodies. Afterthat the samples were fixed with 1% formaldehyde and stored at 4°C for atime no longer than 48 hr until flow cytometry acquisition.

Flow Cytometry Acquisition and Intracellular Cytokine Analysis in CD3+ and CD14+ CellsIntracellular cytokine production was evaluated with three-color (lympho-

cytes) or two-color (monocytes) flow cytometric analysis using a FACSCaliburcytofluorimeter (BD, San Jose, CA, USA) with CellQuest software for datastorage and analysis. In each cell suspension, 20,000 (lymphocytes) or 40,000events (monocytes) were measured on a FACSCalibur flow cytometer. Wedecided to investigate cytokine production according to the instructions pro-vided by Schuerwegh et al. (2003). Briefly, CD3+ cells were defined and gatedon a dot-plot of CD3-PerCP versus side scatter. Within this gate, intracellularcytokine-producing cells were determined in the next dot plots of CD3-PerCPvs. measured cytokine-PE and CD8-FITC vs. measured cytokine-PE. A similarprocedure was conducted in the case of cells stained with CD14-FITC. In thecase of intracellular cytokine staining in lymphocytes, the percentage ofCD3+CD69+ cells, typically equal to or greater than 95% in all examinedcases, was accepted as a positive control of activation. Examples of cell gatingand cytokine analysis are presented in Figures 1 and 2.

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Figure 1: Representative dot-plots showing intracellular cytokine staining in T cells. T lympho-cyte cell gating (SSC vs. CD3-PerCP) preceded the analysis of the intracellular cytokinestaining (Fig. 1A). The next dot-plots show intracellular cytokine analysis of TNF-alpha (Fig 1B1),IFN-gamma (Fig. 1C1), and IL-8 (Fig. 1D1) staining in CD3+ cells. Other dot-plots analyzedintracellular cytokine staining of TNF-alpha IFN-gamma and IL-8 in CD3+CD8- and CD3+CD8+(Figs. 1B2, C2, and D2, respectively) cells based on prior CD3+ gating. In parentheses are thepercentages of the cells expressing cytokines. Localization of the co-expression of CD3+ andits subpopulations (CD3+CD8- or CD3+CD8+) vs. the analyzed cytokine (TNF-alpha, IFN-gamma, and IL-8+) was performed based on appropriate isotype control localization (datanot shown).

C3

SS

C

CD8-FITC

TNF-

alph

a-P

E

TNF-alpha-PE

A)

D1) D2)

C2)

C1)

B1)

CD3 + TNF-alpha +(65.6%)

SSC.vs CD3-PerCP

CD3 + IL-8 +(10.8%)

CD3 + CD8-IL-8 +(6.3%)

CD3 + CD8-IFN-gamma +(21.5%)

CD3 + CD8 + IFN-gamma +(14.3%)

CD3 + IFN-gamma +(35.8%)

CD3 + CD8-TNF-alpha + (44.1%)

CD3 + CD8 + TNF-alpha +(20.3%)

CD3 + CD8 + IL-8 +(4.5%)

B2)C

D3-

Per

CP

CD

3-P

erC

P

CD

3-P

erC

P

CD3-PerCP

IL-8-PE CD8-FITC

IL-8

-PE

CD8-FITC

IFN

-gam

ma-

PE

IFN-gamma-PE

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Figure 2: Representative dot-plots showing intracellular cytokine staining in CD14+ cells. Theanalysis of the intracellular cytokine staining preceded CD14+ cell gating (SSC vs. CD14-FITC)(Fig. 2A). The next dot-plots show intracellular cytokine analysis of TNF-alpha (Fig. 2B), IL-6 (Fig.2C), IL-10 (Fig. 2D), MCP-1 (Fig. 2E), and IL-8 (Fig. 2F) in CD14+ cells. In parentheses are thepercentages of the cells expressing cytokines. Localization of the co-expression of CD14+ vs.the analyzed cytokine (TNF-alpha, IL-6, IL-10, MCP-1, and IL-8+) was performed based onappropriate isotype control localization (data not shown).

TNF-

alph

a-P

E

SS

C

CD14-FITC

B)

A)

E)F)

D)

C)

CD14 + IL-6 +

(92.4%)

CD14 + IL-10 +

(35.3%)

CD14 + MCP-1 +

(61.9%)CD14 + IL-8 +

(99.9%)

SSC VS. CD14-FITC

CD14 + TNF-alpha +

(38.5%)

IL-6

-PE

CD14-FITC

CD14-FITC

CD14-FITCCD14-FITC

IL-8

-PE

MC

P-1

-PE

IL-1

0-P

E

CD14-FITC

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Statistical AnalysisCalculations and statistical analyses were performed using MS Excel 2000

(Microsoft Inc., USA) and Statistica version 6.0 (StatSoft Inc., Tulsa, OK,USA) for Windows software. The Shapiro-Wilk test was employed to investi-gate the data distribution. Because the data distributions varied, appropriatestatistical tests (parametrical or non-parametrical) were employed to comparedifferences between the groups. Analysis of intergroup differences was per-formed using one-way analysis of variance (ANOVA) or the Kruskal–Wallistest. Individual study groups were compared using the Student t-test orMann–Whitney U-test. The Bonferroni procedure (Dunn’s multiple compari-son procedure) was employed when more than 2 groups were compared.A p-value <0.05 was considered statistically significant.

RESULTS

Immunophenotypic AnalysisStatistical analysis of the percentages of the cell populations in the total

group of patients (endometriotic patients compared with the control group)showed no significant differences (Table 1). The mean CD3+CD4+/CD3+CD8+ratio (mean ± SEM) was insignificantly lower in endometriosis patientscompared with the control group (2.3 ± 0.2 vs. 2.8 ± 0.5, respectively). Thepercentages of the analyzed cell populations in both groups of patients werenot dependent on menstrual phase cycle (data not shown). When theendometriosis group was divided with regard to the severity of the disease, nosignificant differences in the percentages of the cell populations in mildendometriosis compared with advanced were noted (Table 1). We did notperform an analysis of the mean fluorescence intensity (MFI) in theCD14+HLA-DR+ cell populations because of the different clones of anti-human HLA-DR antibody used in the cell staining.

Intracellular Cytokine Production in CD3+ CellsThe percentage of activated lymphocytes with cytokine expression varied

depending on the cytokine. In general, within T lymphocytes, both in womenwith endometriosis and in the control group, the highest percentage of cellsexpressed TNF-alpha, a lower level was noted in the case of IFN-gamma,and the lowest for IL-8 (Figure 3A). No significant differences in the percent-ages of CD3+, CD3+CD8-, and CD3+CD8+ cells stained for any of the inves-tigated cytokines were observed (Figures 3A, 3B, and 3C). The percentagesof CD3+ and CD3+CD8- cells stained for TNF-alpha or IFN-gamma werelower in endometriosis patients compared with the control group (Figures3A and 3B). In contrast, the percentages of CD3+ and CD3+CD8- cells with

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intracellular expression of IL-8 were higher in the endometriosis patientscompared with the control group (12.2 ± 0.8 and 8.1 ± 0.6 vs. 9.4 ± 1.1 and6.2 ± 0.7, respectively) (Figures 3A and 3B). There were no observed differ-ences in intracellular staining for TNF-alpha, IFN-gamma, or IL-8 inCD3+CD8+ cells when women with endometriosis were compared with thecontrol group (Figure 3C). After dividing the endometriosis group accordingto severity of disease, post hoc analysis of the individual comparisonsshowed a significantly lower percentage of CD3+CD8- cells stained for IFN-gamma (p = 0.0045) in women with advanced endometriosis compared withthe control group (17.4 ± 1.6 vs. 25.5 ± 2.0) (Figure 4B). Prior to our study wealso tried to calculate the methodology of the intracellular cytokine stainingof IL-10 and IL-6 in stimulated CD3+ cells and their subpopulations; how-ever, using the stimulation protocol described in the Materials and Methodssection, we were not able to induce production of these cytokines in theexamined cells (data not shown).

Intracellular Cytokine Production by CD14+ CellsThe highest percentages of CD14+ cells were noted in staining for IL-6

and IL-8 (>90%); however, this finding was independent of the presence orabsence of any disease (Figure 3D). In the case of CD14+ cells stained forTNF-alpha and MCP-1, higher values were noted in women with endometrio-sis compared with the control group (42.3 ± 2.8 and 61.9 ± 2.4 vs. 36.7 ± 4.4and 55.4 ± 3.8, respectively); however, no significant differences were observed

Table 1: Analysis of percentage of CD3+, CD3+CD4+, CD3+CD8+, CD3+CD69+, CD16+, CD14+ and CD14+HLA-DR+ in women with endometriosis compared with control group.

Cell population

Groups of patients

Control group(leiomyoma)

(n = 12)

Endometriosis

Endometriosis(total group;

n = 36)

Mildendometriosis

(n = 13)

Advancedendometriosis

(n = 23)

CD3+* 37.5 ± 3.2 39.5 ± 2.2 39.0 ± 4.4 39.8 ± 2.4CD3+CD4+** 51.6 ± 4.1 50.9 ± 2.4 46.7 ± 3.8 53.3 ± 3.1CD3+CD8+** 21.9 ± 2.5 25.1 ± 1.5 23.6 ± 2.5 25.9 ± 1.8CD3+CD69+** 0.8 ± 0.1 0.8 ± 0.1 0.7 ± 0.1 0.8 ± 0.1CD16+* 6.5 ± 1.3 9.2 ± 0.9 7.1 ± 1.0 10.4 ± 1.2CD14+* 11.6 ± 2.1 10.6 ± 0.9 10.7 ± 1.9 10.6 ± 1.0CD14+HLA-DR+*** 96.4 ± 1.0 95.2 ± 0.6 94.7 ± 1.2 95.5 ± 0.6

* Among all isolated mononuclear cells.** Among all CD3+ cells.*** Among all CD14+ cells.All values are expressed as mean percentage (%) ± standard error of the mean (SEM).

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(Figure 3D). There were no differences noted in the intracellular expressionsof IL-6, IL-10, and IL-8 when endometriosis patients were compared with thecontrol group (Figure 3D).

When the endometriosis group was divided with regard to severity of dis-ease, post hoc analysis revealed a significantly higher percentage of CD14+cells stained for MCP-1+ in the women with advanced endometriosis com-pared with the mild endometriosis and control groups (66.9 ± 2.1 vs. 52.9 ± 4.5and 55.4 ± 3.8, p = 0.0032 and 0.0072, respectively) (Figure 4D)

Figure 3: Intracellular cytokine staining of TNF-alpha, IFN-gamma, and IL-8 in peripheral CD3+cells (Fig. 3A), CD3+CD8- (Fig. 3B), and CD3+CD8+ (Fig. 3C) cells, and TNF-alpha, IL-6, IL-10,MCP-1, and IL-8 in peripheral CD14+ cells (Fig. 3D) in women with endometriosis (wholegroup), and the control group (uterine leiomyoma patients). The number of analyzed sampleswas for lymphocytes (women with endometriosis: n = 25; control group: n = 11) and for mono-cytes (endometriosis patients: n = 36; control group: n = 12). All values were presented asmean ± SEM.

control group

A)

D)C)

B)

endometriosis

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DISCUSSION

Data published until now show that endometriosis may be considered a localdisease with systemic subclinical manifestations. This is supported by the

Figure 4: Intracellular cytokine synthesis of TNF-alpha, IFN-gamma, and IL-8 in peripheral CD3+cells (Fig. 4A), CD3+CD8- (Fig. 4B) and CD3+CD8+ (Fig. 4C) cells, and TNF-alpha, IL-6, IL-10,MCP-1, and IL-8 in peripheral CD14+ cells (Fig. 4D) of women with endometriosis with regardto the stage of disease (mild endometriosis, rASRM I-II, and advanced endometriosis, rASRMIII-IV). The number of analyzed samples was for lymphocytes (rASRM I-II: n = 6; rASRM III-IV: n =19; control group: n = 11) and for monocytes (rASRM I-II: n = 13; rASRM III-IV: n = 23; controlgroup: n = 12). All values were presented as mean ± SEM. *p < 0.05 compared with the controlgroup using post hoc analysis with the independent samples t-test and Bonferroni procedure.**p < 0.05 compared with the control group and mild endometriosis using post hoc analysiswith the independent samples t-test and Bonferroni procedure.

control group

A) B)

C)

mild endometriosis (ASRM: I-II)

advanced endometriosis (ASRM: III-IV)

*

**

D)

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observation of aberrant serum levels of CRP, SAA, TNF-alpha, MCP-1, andIL-8 (Agic et al., 2006). In contrast, the percentage of leukocytes at thesystemic level in women with endometriosis seems to be unchanged. Althougha study by Gagne et al. (Gagne at al., 2003) showed increased percentages ofperipheral CD3+ and CD14+HLA-DR+ and Szyllo et al. (Szyllo et al., 2003)noted a decreased percentage of peripheral CD16+ and an increased CD4+/CD8+ ratio in women with endometriosis, other studies, including ours, do notsupport these findings (Oosterlynck et al., 1994; Kusume et al., 2005). Incontrast, systemic cell activity in women with endometriosis remains anunresolved question. It is unknown whether the defect (if any) in peripheralimmune cells activity is intrinsic in nature or a consequence of endometriosisprogress.

In this study, to examine immune cell activity we used the intracellularcytokine staining assay (ICS), which is a commonly accepted method for cytok-ine production studies. In this assay, the measurement of cytokine expressionis followed by cell stimulation in vitro and disruption of cytokine excretion(Pala et al., 2000). Generally it is assumed that stimulation of cells does notchange the cytokine profile, but only intensifies cytokine gene expressions,making possible an easier detection of cytokine production by the intracellularcytokine staining assay (Cohen et al., 1999). More recently, positivecorrelation between intracellular and extracellular cytokine production afterLPS (IL-1β, IL-6, IL-10, and IL-12) and PMA and ionomycin stimulation (IL-2and IFN-γ) in vitro has been shown (Schuerwegh et al., 2003). It is tempting tospeculate whether there is any relationship between cellular and serumcytokine levels (Jason et al., 2001). Grob et al. (2003) noted significantlyincreased intracellular cytokine levels of IL-8 in CD4+ and CD8+ lymphocytesof patients with allergic asthma, but at the same time they observed non-significantly altered IL-8 serum levels in the investigated group of patientscompared with controls. For this reason we focus our discussion on comparingour results with those where cell culture assays were employed.

In the women with advanced endometriosis we observed a decreased per-centage of IFN-gamma-producing CD3+CD8- cells compared with the controlgroup. In contrast, we did not observe any significant differences in intracellu-lar deposits of IFN-gamma in the whole population of CD3+ cells. This is inaccord with Szyllo et al. (2003), who observed no significant difference inintracellular deposits of IFN-gamma in the T cells of women with endometrio-sis. It is interesting that in supernatants collected after PHA stimulation ofPBMCs they noticed a significant decrease in the concentration of IFN-gamma. We were unable to find any significant differences in intracellularcytokine deposits of TNF-alpha, IL-6, IL-10, and IL-8 in the investigated cellpopulations. Szyllo et al. (2003) observed a significantly higher percentage ofperipheral CD3+ cells stained for TNF-alpha in women with endometriosiscompared with a control group. They also noted in women with endometriosis

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significantly and insignificantly increased percentage of CD3+ with intracel-lular expression of IL-6 and IL-4 respectively, not investigated in our study.

In turn, an in vitro study by Braun et al. (Braun et al., 1996) failed toshow any significant differences in IL-6 and IL-8 production by LPS-stimulated monocytes of women with endometriosis. However, they showedsignificantly higher TNF-alpha production in a 24-hr LPS-stimulated mono-cyte cell culture, which contrasts with the outcome of our study showing nodifferences in the percentage of monocytes stained for TNF-alpha after 9 hr ofstimulation in vitro. However, similar to our observations, IL-10 productionby LPS-stimulated monocytes was unaffected in their study (Braun et al.,1996). The explanation of the partial difference between our results and thoseof Braun et al. (Braun et al., 1996) could be related to the time of cell culture(9 hr vs. 24 hr) and the kind of cell culture suspension (heterogeneous vs.homogenous).

The unresolved question concerning women with endometriosis is thecytokine-related type of response at the systemic level. The increased concen-tration of IL-4 at the mRNA and protein levels observed in the peripheralblood of women with endometriosis (Hsu et al., 1997) could indicate the Th2-type immune response. This concept supports outcomes from our study show-ing increased intracellular expression of MCP-1 in CD14+ cells. It is believedthat chemokine expression may be essential in the regulation of the Th1/Th2type response. The expressions of MIP-1α, MIP-1β, and RANTES correlatewith the Th1-like phenotype (Schrum et al., 1996); in turn, the presence ofMCP-1 enhances the production of IL-4 (Karpus et al., 1997). More recently,Omata et al. (Omata et al., 2002) observed that the production of IFN-gammais decreased in naïve T cells stimulated with MCP-1-primed dendritic cells(DCs).

This outcome indicates that MCP-1, through DCs, may inhibit Th1 celldevelopment. In this study we observed increased intracellular cytokinedeposits of MCP-1, which could be responsible for the Th2-like responsepolarization observed mainly in advanced endometriosis. The notion of theprevalence of the Th2-type response at the systemic level in women withendometriosis was recently supported by Antsiferova et al. (Antsiferova et al.,2005), who showed increased intracellular levels of IL-4 and IL-10 in periph-eral lymphocytes. Endometrosis is considered as a disease with an autoim-mune background (Nothnick, 2001). It is interesting that a high rate ofautoimmune diseases was noted among women with endometriosis (Sinaii et al.,2002); however, the mechanisms of autoimmune modulation in these womenare poorly investigated and still awaiting exploration.

In our study we also observed a decreased percentage of CD3+CD8- cellsstained for IFN-gamma. The population of CD3+CD8- cells is considered bysome authors to be CD4+ cells owing to the dramatic decrease in CD4 expres-sion after PMA stimulation (Faas et al., 2000). However, this may be doubtful,

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because among CD3+CD8- cells may be both NK cells and/or “double-nega-tive” T cells (Baran et al., 2001). For this reason it is difficult to state exactlywhich cell population (NK cells or CD4+ cells) possesses a decreased ability forIFN-gamma synthesis.

Taken together, the results of our study show that peripheral mononu-clear cells derived from women with advanced stages of endometriosis havedifferent intracellular cytokine synthesis in vitro compared with control cells.This observation may favor the concept that functional abnormalities inperipheral lymphocyte and monocyte cytokine production (if there are any)are more a consequence than a cause of endometriosis. In contrast, our resultsdo not support the hypothesis about differential cytokine synthesis at thesingle-cell level by T cells and monocytes of women with mild endometriosiscompared with those without endometriosis. The increased intracellularsynthesis of MCP-1 in monocytes presented here could provide indirect prooffor the Th-2 type response polarization in women with endometriosis, which ismainly observed in advanced stages of this disease. This may support thetheory which considers endometriosis as a disease with an autoimmunebackground by presenting evidence at the systemic level.

ACKNOWLEDGMENTS

The study was supported by grant No. 3P05E 077 24 from the Polish Ministryfor Scientific Research and Information Technology and partly by a statutorygrant of the Laboratory of Reproductive Immunology at Institute of Immunologyand Experimental Therapy (No. 17/2005).

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