The role of the atypical chemokine receptor D6 in the placenta

D6 is an atypical chemokine receptor related to CCR1-5 that binds to many inflammatory CC chemokines. Experiments using transfected cell lines have shown that upon binding to a chemokine ligand D6 does not trigger cellular signalling pathways, but rather acts to scavenge the bound ligand. It achieves this by constitutively travelling to and from the cell surface via early and recycling endosomes, internalising chemokines bound when it is at the cell surface. Over time, D6 removes a large amount of ligands from the extracellular compartment. In vivo, this scavenging activity is thought to regulate the level of CC chemokines, and thus controls inflammation locally and systemically. Lack of D6 has been shown to result in elevated amounts of bioavailable chemokines, and is associated with over exuberant inflammatory responses. In human, D6 mRNA and protein is highly expressed in trophoblast-derived gestational tissues. The expression of D6 mRNA in the placenta is by far the highest, compared to other solid tissues being studied. The importance of D6 in protecting the offspring has been demonstrated in animals. In pigs, a defect in D6 expression was discovered in placental attachment sites in endometrium from arresting fetuses. In mice, lack of D6 results in an increase in fetal loss after challenge with lipopolysaccharide (LPS) or antiphospolipid autoantibodies (aPL), and an increase in the number of abnormal pups when mouse embryos are transferred into fully allogeneic pseudo-pregnant female recipients. In view of these results suggesting a critical role for D6 in placental mediated complications, the expression and molecular function of D6 in primary human trophoblast cells were studied, as to date in vitro human studies have utilised the choriocarcinoma cell line BeWo or immortalised cell lines engineered to over-express exogenous D6. Secondly the impact of D6 deficiency on placental structure, chemokine expression and leukocyte abundance in mice was examined. Chapter 3 presents the results of experiments on primary human trophoblasts. Protocols for routine primary trophoblast isolation, purification and culture from fresh term placentas were optimised in our laboratory. D6 mRNA was detected in these primary cells. Using Western blotting, immunofluorescence and flow cytometry, D6 was shown to be present predominantly in the intracellular vesicles of the cells. Competition chemokine uptake assays, analysed by flow cytometry, showed that CCL2 was internalised by trophoblasts using D6. Competitive chemokine scavenging assays, analysed by quantitative Western blot, confirmed that D6 was functioning as a chemokine scavenger on primary human trophoblasts and that it progressively removed substantial quantities of chemokine from medium bathing the cells. This is the first set of experiments that confirms D6 is present, and functioning as a chemokine scavenger in primary human cells. Chapter 4 contains the results from the mouse experiments. Even in an unchallenged environment it was shown that, on the DBA-1 genetic background, D6 deficiency in the mother and pups leads to higher rates of stillbirth and neonatal deaths, resulting in a reduction in the number of pups weaned per litter than their WT counterparts. By gestational age E14, pup weight was significantly smaller in the D6 KO mice. Using stereological techniques, the placenta of the D6 KO mice at this gestation was found to have a smaller labyrinthine zone. The volume of the labyrinthine zone was positively correlated with pup/placenta ratio. These phenotypes could be due to a maternal or fetal effect of D6 deficiency. To ascertain the answer to this question, the experiment at E14 was extended by breeding DBA-1 females heterozygous for the deleted D6 allele (D6 HET) with D6 deficient (D6 KO) males. In this model the phenotypes of D6 KO pups and placentas could be compared with their D6 HET siblings that developed in a mother expressing some D6 (i.e. D6 HET). Although there were no differences in pup weight, placental weight and pup/placenta ratio between these two groups, stereology revealed a decrease in labyrinthine zone volume fraction in the D6 KO placentas in comparison to their D6 HET siblings. The observed fetal compromise and placental defect at E14 was not apparent at the later gestational age of E18. Luminex multiplex protein assay showed an elevated level of circulating chemokine CCL2 in the serum of D6 KO pregnant mice in comparison to their WT counterpart, so loss of chemokine regulation could be responsible for the defects observed in D6 deficient placentas. In summary, D6 deficiency results in an increase in perinatal death, a fundamental defect in placental formation (reduced labyrinthine zone) and dysregulation of circulating chemokine levels. Chapter 5 discusses the mechanisms of D6 in regulating placental formation and reproductive outcome and the novel insights that this work provided into placental D6 function. It also describes the design of future experiments to reveal the precise role of D6 in chemokine regulation and cell signalling in reproductive immunology, and discusses how D6 might contribute to pregnancy outcome in humans