1 Department of Obstetrics and Gynecology and 2 Department of Medical Research, College of Medicine and the Hospital, National Taiwan University, 7 Chung-Shan South Road, Taipei, Taiwan
3 To whom correspondence should be addressed. Email: hnho{at}ha.mc.ntu.edu.tw
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Abstract |
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Key words: adenomyosis/endometrium/killer cell inhibitory receptor
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Introduction |
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The decreased NK cell activity in peripheral blood and peritoneal fluid of women with endometriosis has been well established in recent years (Oosterlynck et al., 1991; Ho et al., 1995
). It is thought to promote implantation of the endometrium as a tissue graft (Lefkowitz et al., 1988
), and its cause is probably due to overexpression of KIRs. Our previous study demonstrated that increased expression of NKB1 and EB6 was found on NK cells in peritoneal fluid in women with advanced stage endometriosis (Wu et al., 2000
). Another study showed a similar result, where the proportion of KIR2DL1+ NK cells was increased in peritoneal fluid and peripheral blood in women with endometriosis (Maeda et al., 2002
). Moreover, the endometriotic tissue could also affect NK cells by an unknown mechanism to impair the NK cytotoxicity. Our previous studies demonstrated that NK cytotoxicity in endometriosis could be affected by either cytokines or T cells (Ho et al., 1996a
, 1997
). The KIRs expressed on T cells might also play a role in the regulation of NK cytotoxicity.
In contrast to endometriosis being characterized by ectopic endometrium in the peritoneal cavity, adenomyosis is defined as the presence of endometrial glands within the myometrium. The only difference between adenomyosis and endometriosis is the site of ectopic endometriotic tissues, i.e. within or outside of the uterus. In this study, we tried to measure different kinds of KIR expression on NK and T cells in different parts of the uterus, as well as different expression of KIRs between women with and without adenomyosis, in an attempt to find the possible role of KIRs in the development of adenomyosis.
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Materials and methods |
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Peripheral venous blood, myometrium and endometrium were obtained immediately after the uterus was removed from the women in both groups. In the study group, the myometrium was acquired from the tissue where there is coarsely trabeculated and diffusely hypertrophied myometrium stippled with foci of ectopic endometrium, while in the control group, the myometrium was obtained from tissue other than uterine myoma. This tentative grouping determined by the naked eye was then found to be fully consistent with the final diagnosis provided by the pathologists. The cervical tissue was only derived in women without adenomyosis.
The aspirated blood was collected in glass tubes containing heparin, and was processed within 30 min. Peripheral blood mononuclear cells (PBMCs) were isolated by layering over Ficoll-Paque (Amersham Pharmacia Biotech, Uppsala, Sweden) and centrifuging at 800 g for 20 min. The isolated PBMCs were washed twice with RPMI-1640 medium (Life Technologies, Inc., Grand Island, NY) to remove residual Ficoll-Hypaque solution, and were reconstituted to a final cell concentration of 12 x 106 cells/ml. The viability of PBMCs was verified with a trypan blue exclusion test. Viability was typically more than 95%.
Myometrial, endometrial and cervical tissues were collected aseptically and separately in a tissue flask containing RPMI-1640. Contaminated blood was removed by washing with RPMI-1640. Tissues were cut into tiny pieces (0.5 mm3) with a surgical knife, and were suspended in 5 ml of RPMI-1640. The suspensions were ground and passed consecutively through different sized mechanical sieves (sieve size 150, 300 and 400), and were overlaid on discontinuous (100%/50%/30%) Percoll gradients (Sigma Chemical Co., St Louis, MO). After centrifuging at 800 g for 45 min, mononuclear cells were obtained from the interface of 100 and 50% Percoll solution, and were then reconstituted to a final concentration of 12x106 cells/ml. The viability of mononuclear cells was verified with a trypan blue exclusion test. Viability was typically more than 90%.
Immunophenotypic analysis with three-colour flow cytometry
The methods have been described in detail previously (Yang et al., 2000). In brief, monoclonal antibodies (mAbs) conjugated with fluorescein isothiocyanate (FITC) or phycoerythrin (PE) were obtained (Becton Dickinson, San Jose, CA). Mononuclear cells were incubated with mAbs at 4°C for 30 min and then were washed twice in phosphate-buffered saline (PBS) containing 2% fetal calf serum (FCS) and 0.1% sodium azide. These samples were fixed with 0.5% paraformaldehyde. Immunofluorescence and three-colour flow cytometric analyses were done using a FACScan cytofluorimeter (Becton Dickinson) with a computer interface to software (Hewlett-Packard Consort 32, Becton Dickinson) for full-list mode data storage, recovery and analysis.
The following combinations of mAbs were used: FITCanti-CD45/PEanti-CD14 (LeucoGATE), FITCanti-IgG1/PEanti-IgG2a (negative control), FITCanti-CD3/PEanti-CD19 (T cells), FITCanti-CD3/PEanti-CD56 (NK cells), FITCanti-CD56/PEanti-NKB1/PerCPanti-CD3, FITCanti-CD56/PEanti-GL183/PerCPanti-CD3, FITCanti-CD56/PEanti-EB6/PerCPanti-CD3, FITCanti-CD56/PEanti-CD94/PerCPanti-CD3, FITCanti-CD4/PEanti-NKB1/PerCPanti-CD3, FITCanti-CD4/PEanti-GL183/PerCPanti-CD3, FITCanti-CD4/PEanti-EB6/PerCPanti-CD3, FITCanti-CD4/PEanti-CD94/PerCPanti-CD3, FITCanti-CD8/PEanti-NKB1/PerCPanti-CD3, FITCanti-CD8/PEanti-GL183/PerCPanti-CD3, FITCanti-CD8/PEanti-EB6/PerCPanti-CD3 and FITCanti-CD8/PEanti-CD94/PerCPanti-CD3. Leucogate was used to measure the proportion of lymphocytes in the sample being studied without any scatter gates. The gate was set around the lymphocytes (CD45+CD14) to exclude other cells from analysis. The Simultest control (mouse FITCanti-IgG1/PEanti-IgG2a) was used for background control. Doublets, i.e. two cells either stuck together or very close in space, were strictly excluded from the calculation. In each cell suspension, 10000 events in PBMCs as well as 20005000 events in tissue mononuclear cells acquired for gated lymphocytes were measured. The density of surface markers was expressed as the mean fluorescence intensity (MFI) of cells stained with specific mAbs of KIRs.
Statistical analysis
All values are expressed as mean±SD. As the data were not normally distributed, the MannWhitney U-test for non-parametric data was used to compare the difference between the groups. A P-value <0.05 was considered statistically significant.
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Results |
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The mean number of total lymphocytes per g of tissue was 2.6 (range 0.16.5)x104 in the myometrium and 1.9 (0.74.4)x106 in the endometrium in women with adenomyosis, similar to those numbers obtained in the myometrium [1.8 (0.35.4)x104] and endometrium [2.5 (0.17.5)x106] in women without adenomyosis. The mean lymphocyte number in the cervix was 1 (0.33.4)x104 in women without adenomyosis.
Among myometrium, endometrium and cervix in women without adenomyosis, there was no difference in the fraction of NK and T cells. The MFI of CD94 was significantly higher in the subpopulations of CD56+ (114.5±99) and CD56+CD94+ (175.1±113) cells in the endometrium, compared with those in the myometrium (38.5±29.9 and 94.2±48.5) and cervix (44.6±33.8 and 84.8±38). The MFI of GL183 in the CD56+GL183+ subpopulation in endometrium was 157.4±97.8, much higher than that in the cervix (80.9±52). On the other hand, NKB1 and EB6 were similarly expressed on NK cells in different parts of the uterus (Table I). The difference was also not prominent in the expression of various kinds of KIRs on CD4+ and CD8+ T cells in different compartments of the uterus (Table II).
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Discussion |
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Although heterogeneity in expression of KIRs between blood and uterus was found, there has not been any report describing KIR expression in different parts of the uterus. Our results revealed that there was an increased CD94 expression on NK cells in the endometrium compared with that in the myometrium and cervix. This might imply that the NK activity is by nature depressed in the endometrium compared with that in the myometrium and cervix because the increased expression of KIR generally represented a decreased NK activity (Wu et al., 2000). This naturally depressed NK cytotoxicity in the endometrium might therefore be one of the possible mechanisms that account for the development of endometriosis and/or adenomyosis in women of reproductive age. Without performing functional studies using class I HLA-expressing target cells, however, we cannot confirm that the increased expression of CD94 could inevitably result in inhibition of NK activity in this study.
We also demonstrated decreased expression of NKB1 and GL183 on endometrial NK cells in women with adenomyosis compared with that in women without adenomyosis. It may be a compensatory effect, in which the NK cytotoxicity is activated in women with adenomyosis in order to eradicate the abnormal endometrial cells that might exit the eutopic site of the endometrium. It also implies that the abnormal endometrial cells, rather than the impaired NK cell function, account for the development of adenomyosis. The different immunological expression of KIRs in the eutopic endometrium is supported by a previous report (Braun et al., 2002), in which a reduction in apoptosis of endometrial cells was found in the eutopic endometrium in women with endometriosis due to reduced macrophage trafficking into the eutopic endometrium. However, GL183 recognizes both inhibitory and activatory forms of KIRs, which could result in either inhibition or activation of the NK-mediated cytolytic activity (Moretta et al., 1995
). Since western blotting and reverse transcription (RT)PCR were not performed in this study, the possibility remains that inhibitory as well as activatory forms of GL183 may be decreased. Similarly, an alternative explanation for the decreased NK cytotoxicity seen in endometriosis is that there may be decreased expression of activatory receptors rather than increased inhibitory KIR expression. As K562 target cells generally lack class I MHC expression (Rouas-Freiss et al., 1997
), any changes in the levels of inhibitory or activatory KIRs should not necessarily affect NK cytotoxicity once K562s were employed as the target cells.
Our results did not reveal different KIR expression on the myometrial NK cells between women with and without adenomyosis. This unlike the finding of an increased KIR expression on the peritoneal NK cells in women with endometriosis (Wu et al., 2000; Maeda et al., 2002
). As a result, the local immunological appearance in response to the ectopic endometrium might be different between adenomyosis and endometriosis.
Unlike the different expression of KIRs on NK cells, we demonstrated that KIRs were expressed similarly on both CD4+ and CD8+ T cells among various uterine tissues and between women with and without adenomyosis (Tables II and IV). In agreement with previous reports (Moretta et al., 1996; Mingari et al., 1996
), we also found that KIRs were expressed more frequently on CD8+ T cells than on CD4+ T cells. One reasonable explanation for the advantage of this event is that the CD8+ T cells have NK-like activity, and would thus be deleterious to normal cells if they did not express KIRs. Their defective expression could be involved in autoimmune diseases caused by autoreactive cytotoxic T cells (Mingari et al., 1998
).
In conclusion, we demonstrated a decreased expression of KIRs on NK cells in eutopic endometrium in women with adenomyosis compared with that in women without adenomyosis. It might be a compensatory effect in which the NK cytotoxicity is activated in order to eradicate the abnormal endometrial cells that might exit the eutopic site of the endometrium.
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Acknowledgements |
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References |
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Submitted on November 12, 2003; accepted on May 25, 2004.
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