The highly proliferative and abundant population of CD56bright
NK cells which inhabits the human uterus within days after the LH surge is thought to arise from uterine recruitment of circulating blood and in situ proliferation (2
). Here, we describe significant increases in adhesion of CD56bright
blood lymphocytes taken at the LH surge of fertile females, to L-selectin ligands in decidualized mouse uterus but not to ligands in either LN or PP, indicating a tissue specific effect. Furthermore, lymphocytes from male donors exhibited enhanced adhesive capacity after incubation with E2
, low pre-ovulatory levels of P4
or LH as compared to cell aliquots cultured without additives.
cells constitute less than 1% of peripheral blood lymphocytes and differ in function from the CD56dim
subset by producing greater amounts of IFN-γ, TNF-β, GM-CSF, IL-10 and IL-13 when stimulated by IL-12, 15, 18 or PMA (25
), and by exhibiting reduced natural cytotoxicity in the unstimulated state (26
). Uterine NK cell populations differ from peripheral blood subsets in several species (2
), particularly in terms of gene transcription (29
). In primates, dNK cells express the phenotype and share the cytokine and comparatively non-lytic characteristics of peripheral CD56bright
). It is attractive to postulate that a subset of blood CD56bright
cells homes to the uterus and undergoes terminal differentiation in response to uterine stromal factors such as IL-15 (5
). We speculate that major differences between blood and dNK cells can be attributed to these uterine events, as circulating NK cells are immature.
Migration of leukocytes to inflammatory sites is induced by chemokines, secreted by cells at the site, which bind to seven-transmembrane G-protein couples receptors (34
). Hormonal effects on expression of chemokines and their receptors have been described; E2
induces increased production of inflammatory chemokines in dendritic cells and monocytes (35
). Administration of exogenous estrogens during fertility treatment results in increased numbers of dNK cells and in glandular IL-8/CXCL8 production (36
). Progesterone induces MIG/CXCL9, while E2
inhibits its production (37
). Investigations of menstrual cycle effects on production of chemokines or their ligands reveal up-regulation of endometrial transcripts for CXCR1, CCR5, CCR2B and, to a lesser extent, CXCR4 in the luteal phase (38
) and increased expression of CXCL10 (39
) and CXCL9 but not CXCL11 or CXCL12 (40
) in decidua. Circulating CD56bright
cells express high levels of CXCR3, the ligand for CXCL9, 10 and 11 and moderate levels of CXCR4, the ligand for CXCL12, and demonstrate migration in response to these signals (41
). Thus, this subset is well-equipped to respond to cyclic signals from dendritic and stromal cells in the uterus.
Here, we examined the effect of the menstrual cycle on the functional adhesiveness of peripheral blood lymphocytes, the source of dNK cell progenitors suggested from mouse models. Mouse, rather than human uterine tissue was used as the substrate to eliminate differences in stage of cycle in the uterus, and because the assay requires use of relatively fresh substrate tissue (less than 2 weeks of storage at −20°C). We found significant enhancement of adhesive capacity of lymphocytes only at LH surge and only when decidua was used as substrate. When only CD56bright cells were enumerated, it was apparent that they constituted the majority of adherent cells and thus could be preferentially recruited. Since antibody-blocked lymphocytes showed no alteration in adhesion, any changes in adhesive capacity were mediated by activation of L-selectin, presumably by circulating factors.
Direct response of CD56bright
cells to menstrual cycle hormones is debatable; neither rat nor human lymphocytes express α forms of the receptor for estrogen (ER) or progesterone (PR) (42
) but mouse NK cells do (46
). It is reported that dNK cells express ERβ1, but these are non-circulating, terminally differentiated cells (49
). Our findings of a positive relationship between estrogen and adhesion to decidua, a stronger association between LH levels and adhesion, and a biphasic effect of P4
are suggestive of complex hormonal influences in trafficking potential. It appears that rising E2
prior to the LH surge create a trafficking window enabling CD56bright
cell egress from circulation. After ovulation has occurred, predominantly high P4
levels close the window and CD56bright
cells cease trafficking. A critical role for E2
on NK cell trafficking to the uterus is unlikely since a study of mice genetically devoid of ERα or ERβ display no alteration in recruitment of uNK cells to the pregnant uterus (50
). It has been reported that lymphocytes of pregnant women, as well as of women in the luteal phase of the menstrual cycle express the LH receptor, but at lower levels than found in ovarian tissue (51
). Therefore, it is quite plausible that peripheral blood lymphocytes are directly activated during the LH surge.
The enrichment of CD56bright
NK cells in the uterine environment is well documented (1
). However, the means by which this enrichment occurs is still not understood. Decidua contains no HEV, the usual site of lymphocyte trafficking to lymphoid tissues. Due to frequent histological observations of mouse NK cells transiting decidual arteries, we speculate that these arteries, which thin and elongate as decidualization progresses, are the site of NK cell invasion.
Menstrual cycle variation has also been described in human blood NK cell lytic function(53
) and in number of NK cells per millilitre of blood (55
). A significant increase in the number of CD56+ cells at the peri-ovulatory period reported for 39 women between the ages of 20 and 29 was postulated to be due to LH and not to E2
or testosterone (55
). We observed larger differences in NK cell populations between individuals in our study group than within individual. While there appeared to be an increase in the percentage of CD56bright
subset (relative to the total lymphocyte population) at LH surge in our cohort B of 7 patients between the ages of 25 and 38, the difference was not significant. We did not enumerate the total NK cells/mL blood.
While it is attractive to postulate that adhesion is representative of NK cell recruitment to the decidualizing uterus, this is difficult to prove in women. Loss of this NK cell population with each menstrual cycle indicates that renewal occurs. It is generally accepted, and these data support the hypothesis, that lymphocyte recruitment is from the peripheral blood. It would appear from our work that either a threshold level of E2
or a surge of LH activates adhesion molecules on the dNK precursor cell surface, enabling a single wave of NK cells or precursor cells to leave the circulation and enter the uterus. This is consistent with a model in which NK cells localize to the DB at LH +3, proliferate until approximately LH+12, then start to disappear, as previously described (1
). Furthermore, modulation of NK cell lytic activity by LH and βhCG at physiological levels has been reported, suggesting a direct interaction between the NK cell and the polypeptide hormones (54
). Complete understanding of the migration and role of dNK cells is expected to have broad clinical impact in women’s reproductive health areas such as infertility, gestational complications (i.e. recurrent spontaneous abortion, preeclampsia), and pathologies such as endometriosis.