Proliferation of uterine natural killer cells is induced by human chorionic gonadotropin and mediated via the mannose receptor

Abstract

The endometrial lining of the human uterus contains a population of phenotypically distinct (CD56(bright), CD16(dim)), tissue-specific, natural killer [uterine natural killer (uNK)] cells that play a key role in the establishment of a successful pregnancy. An increase in the number of endometrial uNK cells occurs when the conceptus implants, and there is a further increase during the early stages of placentation. Here, we describe studies that have identified human chorionic gonadotrophin (hCG), a glycoprotein synthesized by the preimplantation conceptus, as a novel regulator of uNK cell proliferation. The impact of hCG on uNK cells was mediated via the mannose receptor (CD206) rather than by the classical hCG/LH receptor that was not expressed. The mannose receptor and hCG were colocalized on the surface of uNK cells, and proliferation did not occur if cells were incubated with deglycosylated hCG or intact hCG in the presence of excess d-Mannose. These novel observations provide new insight into the endocrine-immune dialogue that exists between the conceptus and immune cells within the receptive endometrium, and have implications for the role of uNK cell-trophoblast interactions and pregnancy outcome.

Figure 1. Isolation and characterisation of uNK cells from non-pregnant late secretory phase human endometrium

FACS analysis showing of cells incubated with the isotype-matched negative control (A) or CD56 antibody (B): y axis is forward scatter and x axis is the FITC value. In panels C and D the region represented by M1 indicates the number of cells that are positive for FITC. This is ~ 1% in isotype control (C) and ~ 95 % after incubation with anti-CD56 (D). Panel E (I-IV) shows uNK cells immunostained for CD56 (red) and ToPro (nuclear counterstain, blue), Panel I purified uNK cells stained with mouse anti-human CD56, Panel II, is a high power view of the same cells. Panel III, formalin-fixed section of 1^st trimester decidua, (positive control), Panel IV negative control. Scale bar = 20μm.

Figure 2. Expression of receptors in uNK cells

Panel A. Expression of MR mRNA (203bp, upper panel) was detected in isolated uNK cells from 1^st trimester decidua and late-secretory phase endometrium, GAPDH (205bp,lower) was detected in all samples. Panel B. Expression of LH/hCG receptor (203 bp, upper panel) was not detected in isolated uNK cells from 1^st trimester decidua or late-secretory phase endometrium but as expected was present in a sample of CL, GAPDH (205bp lower) was detectred in all samples. Lane M represents the DNA hyperladder. Lanes 1 to 4 show uNK cells from 1^st trimester decidua, lanes 5 to 7 shows uNK cells from non-pregnant late secretory phase endometrium, lane 8 shows a first trimester decidual sample and lane 9 is a sample of corpus luteum (positive control for LHR). Panels C to H: FACS analysis of uNK cell surface antigen expression in all cases axis y is the PE [phycoerythrin] value and axis x is the FITC [fluorescein isothiocyanate] value; C are unstained cells (D), isotype-matched negative controls for FITC+PE, late-secretory endometrial MACS®–isolated uNK cell sample negative controls FITC (E) and stained for CD56 (F) or MR (G). Panel H shows analysis of cells co-stained for CD56 and MR, note cells positive for both markers in upper right quadrant. One representative data set is shown of nine experiments performed.

Figure 3. Localization of hCG and MR on uNK cells

Staining of purified uNK cells with antibodies directed against MR (green) and/or hCG (red). Yellow staining shows areas of co localisation. Blue shows nuclear counterstain, To Pro. The left hand column shows both MR and hCG staining superimposed, the middle column-MR alone (green channel) and the right hand column hCG alone (red channel). Panels I to III: cells were untreated. Panels IV to VI: cells were incubated with hCG alone (10,000 ng/ml). Panels VII to IX: cells were incubated with hCG plus D-mannose (1mg/ml). Panels X-XII: cells were incubated with deglycosylated hCG alone (10,000 ng/ml).

Figure 4. MTT proliferation analysis of uNK cell proliferation and mass spectrometry (MS) analysis demonstrating effective deglycosylation of hCG

Panel A: Treatment with hCG alone induced proliferation of uNK cells in a dose-dependent manner, with maximal proliferation observed with 10,000 ng/ml hCG treatment (p<0.001) (green bars). Treatment of uNK cells with hCG plus D-mannose (1 mg/ml) did not induce proliferation and this was also the case when cells were incubated with deglycosylated hCG (10,000 ng/ml). a = p<0.001, 2-way ANOVA between groups. * = p<0.05, *** = p<0.001, 1-way ANOVA within group. n = 5 secretory phase endometrial uNK cell samples. Graph B shows MS analysis of glycosylated hCG, intact protein is ~36 kDa (arrow), α subunit is 13 kDa. Graph C shows MS analysis of deglycosylated hCG with the size of protein reduced to ~22 kDa (*), α subunit is 11.5 kDa. Graph D depicts the MS analysis of the deglycosylation enzyme, PNGase alone.