A specialized population of DCs in the uterus?
It is currently thought that different tissues may contain different populations of DCs. Consistent with that idea, we found subtle differences in the MHC II+ CD11c+ DC population in the uterus, uterine draining lymph node and spleen, even in the non-pregnant state. An example is the appearance of CD8+ DCs expressing very low levels of CD11b in the draining node and spleen (compare Figs 1a–4a and 6a). According to some authors,12 there may be T-cell lineage-related precursor DCs in lymphoid tissues, and this may be the source of the cells we have observed. These DCs may also be a subset of DCs that is thought to circulate only within lymphoid tissue. Another example of a difference between uterine and lymphoid tissues is the presence of CD11b− CD4+ cells in the draining lymph node and spleen, but not the uterus. Such cells may represent a subset of plasmacytoid DCs,40 cells that produce type I interferons upon stimulation.
In some tissues, for example the skin,41 there exists a specialized population(s) of DCs whose major duty is to sample and process antigens found in the tissue and then move to draining nodes to present these antigens to T cells. We hypothesize that the presence of CD11b+ CD4+ DCs in the uterus and draining lymph node, but not in the spleen (Figs 1b, 4b and 6b), may indicate such a specialized DC population. In support of this idea, studies in human decidua11 have documented the presence of a highly proliferative immature population of CD4-expressing DCs that were conspicuously associated with uterine NK cells. In our studies, we do not see a large population of pregnancy-induced uterine DCs that were CD4 positive. In contrast we found that, on day 15 of gestation, the uterine population of CD11b+ CD4+ DCs was decreased as compared with the non-pregnant state. Although the disparate observations may be related to differences in mice and humans, it is formally possible that these DCs normally migrate to the uterine draining nodes or down-regulate their CD4 molecules with activation or as pregnancy proceeds into mid to late gestation in mice.
Another candidate for such a specialized DC population is those DCs that are CD11b+ CD8−, and that represent nearly 60% of the DCs in the uterus and 30–40% in the draining node, yet only 5% of the DCs in the spleen. Other studies have also suggested that the level of CD8 expression in uterine DCs is low.18 The level of CD11b decreased the further away these DCs were from the uterus, suggesting the involvement of local (uterine) regulators of development. Moreover, the proportion of DCs that were CD11b+ CD8− was increased on day 15 of gestation in the uterus and uterine draining node, but not in the spleen. Others21 have found a decrease in the proportion of all CD11c+ cells that are CD11b+, suggesting possible decreased detection of CD11b on MHC II− cells.
The mechanism(s) by which the uterus develops and maintains its population of DCs is unclear. While developmental changes including proliferation and death may play an important role in this process, trafficking is also likely to be important. For example, the increased presence of various populations of CD11b+ DCs in the uterus during pregnancy may reflect circulation and retention based on increased expression of CD11b ligands such as members of the intercellular adhesion molecule (ICAM) family in response to local (trophoblast-induced) stimuli.42
DCs: a critical tool for pregnancy-related immune regulation?
Dendritic cells are thought to be involved in both initiation and regulation of immune responses. The developing paradigm is that for most tissues there is heterogeneity in the DC population based on lineage, phenotype and function. This heterogeneity is driven by possibly systemic and also by tissue- and microenvironment-specific growth and other factors. Current thinking also suggests that within a tissue the balance between immunity and tolerance is based on the functional subtypes of DCs present. The idea persists in the literature that pregnancy exists as a state of obligate immune regulation either systemically or at the maternal–fetal interface. We therefore sought to find at least phenotypic evidence of shifts in DC populations in pregnancy as compared with the non-pregnant state. Contrary to current thinking, the only tissue that seemed to contain a lower proportion of CD45+ cells in pregnancy was the spleen. In addition, we did not find decreased proportions of CD11c+ cells in the uterus, uterine draining node or spleen that were positive for MHC II. Moreover, there was not a significant decrease overall in the level of expression of MHC or CD80. This argues against DC-based suppression, as measured by decreased maturity, associated with pregnancy. A caveat to this is the possibility of down-regulation of MHC, or an influx of immature DCs at a discrete time-point early after implantation.43 Although we could have missed a discrete window when DCs express an ‘immature phenotype’, our studies are focused on the homeostasis achieved at mid gestation (after placental remodelling) which is probably critical to continued pregnancy and prevention of premature birth. There may be other developmentally important time-points related to pregnancy, including peri-implantation, parturition, involution and weaning, when DCs of the reproductive tract also deserve to be examined closely, and these will be the focus of future studies.
In comparison with the uterus, the CD11c+ population in the uterine draining nodes expressed higher levels of both MHC and CD80. For us, this finding is consistent with the idea that trauma at the maternal–fetal interface might lead to uptake of fetal cells or cellular debris that could then be processed and presented by uterine node DCs.1,39 It is also consistent with the idea that pregnancy does not obligately depress DC activation or maturation. Thus quiet, but not suppressed DCs are usually in the uterus, and these can be activated and move to the draining node to present antigen to T cells. In these studies, one antigen that could be presented is H-Y,44,45 as it is present on male fetuses as early as the blastocyst stage. However, other developmentally (fetal) specific antigens could also be presented and drive T-cell responses. These antigens might be important in shaping the T-cell response even during syngeneic pregnancies in mice strains, such as BALB/c, not responsive to H-Y.
A potential limitation of these studies is that they were not performed in multiparous C57BL/6 mice, especially in light of older data46,47 suggesting that multiparity induces systemic tolerance to H-Y. In contrast, more recent data1 suggest that multiparity produces immune activation, and this is consistent with the current examination of lymphoid DCs in pregnant mice. The discrepancy in the reported effect of multiparity may be attributable to the number of pregnancies, with tolerance produced in mothers who have had a larger number (8–11) and activation in those who have had a smaller number (2–3), making comparison of DCs and T cells in the two types of mothers potentially interesting. However, it is also possible that this discrepancy is not related to a change in ‘professional’ DC phenotype, but instead related to an increased depot of chimeric non-DC fetal cells that express male antigen, but cause maternal T-cell depletion as a result of insufficient or inappropriate costimulation. The mechanism of tolerance in grand multiparas would then be similar to that found in non-pregnant females given large quantities of resting B cells.48
An important arising assertion is that the DC population in the uterus and uterine draining nodes, while able to process and present antigen at ‘steady state’, does so with the tendency to inhibit T-cell responsiveness and to generate functional tolerance to fetal antigens.5,24 This idea has been somewhat supported by the finding that specific modulation of DCs adoptively transferred to the uterus49 leads to better pregnancy outcome in a model of abnormal pregnancy.
In our model of normal pregnancy in which the mother is exposed to a common fetal antigen, H-Y, we did not specifically examine the presence of so-called ‘plasmacytoid’ DCs, i.e. DCs that are thought, as a result of low levels of MHC and costimulatory molecules, or through activation of regulatory T cells,50 to support immune tolerance. We did, however, note that a possible subset of these cells, expressing CD4 but not CD11b, was actually not expressed in the uterus, and was decreased in the uterine draining nodes at mid gestation. Further, there was no difference in this subset in the spleens of pregnant versus non-pregnant mice. This argues against the idea of such a sentinel tolerizing DC as a critical mechanism to support normal pregnancy.
We also did not observe a decreased (or an increased) proportion of DCs expressing CD8 but not CD11b, which are thought to support Th1 responses (reviewed for example in51), consistent with another study based on careful immunohistochemistry.21 Moreover, we observed a pregnancy-related increase in DC expression of the chemokine CCL6, which is thought to support inflammatory responses.34 Finally, DC activation is apparent when pregnant mice undergo LPS-induced preterm delivery (P Bizargity, M Phillippe, R Del rio Guerra and EA Bonney, unpublished data).
Classic self–non-self models of immune activation would suggest that, unless specifically suppressed or deviated, the immune system must respond to that which is non-self. Such models would demand comparative study of DCs from the reproductive tracts of female mice mated to genetically similar (for example syngeneic) and dissimilar males, or, in this study, compel examination of tissue surrounding male versus female concepti. Unfortunately, the molecular nature of `non-self', or how DCs might specifically detect it, has only partially been determined with regard to infectious agents.52 It remains unclear how a DC, in the process of presenting a particular fetal antigen, could determine that the antigen came from a syngeneic or allogeneic fetal cell.
However, there exists an alternative model, where metabolic dysregulation or other dysfunction,53 detected at a molecular level,54 is the primary driver of immune system activation. This model, in the context of pregnancy,55 supports study of the steady state of any healthy pregnancy, such as presented herein, where fetal antigen can be recognized.
We did, however, observe an increase in the proportion of DCs that express CD11b, and the majority of this increase appeared to be in CD11b+ cells that are CD4 negative. While the increase in the proportion of cells expressing CD11b suggests an increased capacity to support Th2 responses,51 the implication of the lack of CD4 is unclear and may have to do with increased activation or trafficking. Other studies have also suggested an increased expression of IL-10 over IL-12 in DCs of the uterus;18 however, this was found in pregnant and in non-pregnant mice, suggesting that this ratio, to an extent, may support normal functioning of the uterus apart from pregnancy.56 Finally, in light of other studies,3,57 these data do not suggest that successful pregnancy depends critically on a shift to this class of response.
DCs and tissue homeostasis
Current thinking about DCs suggests that they comprise a diverse, highly plastic and multifunctional group of cells that are at once sensitive to, and eminently capable of shaping, their environments. If this is the case, the functions of DCs may have implications outside that of immune regulation, particularly in a tissue or an animal undergoing significant developmental change. Our observation that uterine DCs can express IL-15 may be an example, although our studies may have missed an early-pregnancy surge in IL-15 expression. In the mouse uterus, NK cells, through expression of interferon (IFN)-γ and other factors, orchestrate several processes including modification of decidual arteries and movement of trophoblast. The function of NK cells in this regard is dependent on IL-15.37,58 As another example, NK cell activity at the maternal–fetal interface may include elaboration of IL-10 which can regulate angiogenesis56 as well as apoptosis,59 and recent data (for example60) suggest that the interaction between NK cells and DCs is important in expression of this cytokine by NK cells. Thus, not only immune function, but also anatomic development at the maternal–fetal interface may be linked to collaboration between NK cells and DCs.
An expanded view, then, is that DCs probably interact, according to a complex scheme, within the uterus, within the draining nodes and systemically in order to support both the development and immune protection of the fetus. The details of the complex signals produced by DCs and their changing capacity to receive signals from their environment deserve wider consideration other than as part of obligate mechanisms of immune tolerance.