Protection against inflammation- and autoantibody-caused fetal loss by the chemokine decoy receptor D6

Abstract

Fetal loss in animals and humans is frequently associated with inflammatory conditions. D6 is a promiscuous chemokine receptor with decoy function, expressed in lymphatic endothelium, that recognizes and targets to degradation most inflammatory CC chemokines. Here, we report that D6 is expressed in placenta on invading extravillous trophoblasts and on the apical side of syncytiotrophoblast cells, at the very interface between maternal blood and fetus. Exposure of D6-/- pregnant mice to LPS or antiphospholipid autoantibodies results in higher levels of inflammatory CC chemokines and increased leukocyte infiltrate in placenta, causing an increased rate of fetal loss, which is prevented by blocking inflammatory chemokines. Thus, the promiscuous decoy receptor for inflammatory CC chemokines D6 plays a nonredundant role in the protection against fetal loss caused by systemic inflammation and antiphospholipid antibodies.

Fig. 1.

D6 expression in human placenta. First-trimester placenta sections were stained with an anti-D6 mAb (A, C, and E) or an irrelevant antibody (B). Invading extravillous trophoblasts were identified by using an anti-cytokeratin 7 mAb (D). (Scale bars, 20 μm.) (E) Low magnification (×250) of D6 staining on syncytiotrophoblast cells. Results are representative of independent experiments performed on at least three individuals.

Fig. 2.

D6 expression and function in trophoblast cells. (A) RT-PCR analysis of D6 and β-actin expression in choriocarcinoma (BeWo, JAR, and JEG-3) and trophoblast (HTR8) cell lines. Ctrl−, non-retrotranscribed RNA. Ctrl+, hD6/pcDNA6 plasmid. Results obtained in one experiment representative of three performed are shown. (B) Confocal microscopy analysis of polarized BeWo cells stained with an anti-D6 (green) and anti-zonula occludens 1 (blue). Propidium iodide (red) was used for nuclear staining. Fifty consecutive confocal scanning images were obtained in the Z stage (step size, 0.2 μm), and three-dimensional reconstruction was obtained as described in Materials and Methods. (B1) Representative sections of the apical and basolateral regions of the cell monolayer. (B2) Blend projection on the XY axes of the three-dimensional reconstruction. Lines define the area considered in the analysis generating the XZ projection shown in B3. (C) D6/HTR8 and CCR5/HTR8 transfectants (black and red lanes, respectively) were stimulated at indicated time point (arrow) with 300 ng/ml CCL3L1, and intracellular calcium concentrations were recorded over time. Results obtained in one experiment representative of three performed are shown. (D) BeWo cells grown to complete confluence on 0.4-μm pore filters were incubated with 10 ng/ml CXCL8/IL-8 (open symbols) or CCL3L1 (filled symbols) added to the upper chamber. At the indicated time points, the chemokine concentration was measured in the upper (solid line) and lower (dotted line) chambers. The dotted line with no symbols represents CCL3L1 concentrations in the upper chamber in the absence of the cell monolayer. (E) BeWo cells (open columns) and D6/CHO-K1 transfectants (filled columns) were incubated with 10 ng of the indicated chemokines per ml. The chemokine concentration in the supernatant was measured after an 18-h incubation. (F) Kinetics of ¹²⁵I-CCL4 scavenging. Mock-transfected (squares) and D6-transfected (circles) HTR8 cells were incubated at 37°C with ¹²⁵I-CCL4 for the indicated time periods. The percentage over total radioactivity input of the trichloroacetic acid (TCA)-soluble (open symbols) and TCA-resistant (filled symbol) fractions recovered in the supernatants are shown. Results in D–F are reported as mean ± SD of triplicate samples of one experiment representative of three performed. SD values are included in the symbol.

Fig. 3.

Role of D6 in LPS- and aPL-induced fetal loss. The percentage of fetal loss (A) and of animals with fetal loss (B) in WT and D6^−/− mice injected with saline (open columns) or LPS (filled columns) is shown. The percentage of fetal loss (C) and of animals with fetal loss (D) in WT and D6^−/− mice injected with saline (open columns) or LPS after treatment with a mixture of blocking antibodies to chemokines (gray columns) or irrelevant antibodies (filled columns) is also shown. The percentage of fetal loss (E) and of animals with fetal loss (F) in WT and D6^−/− mice treated with aPL (filled columns) or IgG from healthy women (open columns) is shown. Numbers inside columns are total numbers of events evaluated. (A, C, and E) Embryonal sacs. (B, D, and F) Injected animals. ∗, P < 0.05; ∗∗, P < 0.01 by Fisher's exact test.

Fig. 4.

Chemokines in the LPS model of fetal loss. (A–E) Serum chemokine concentrations after LPS treatment in WT and D6^−/− male mice. WT (open symbols) and D6^−/− (filled symbols) mice were injected i.p. with 1.35 mg/kg LPS. At the indicated time points, circulating chemokine concentrations were measured by ELISA. Data are from seven mice for each time point. (F–J) Serum chemokine concentrations. WT (open columns) and D6^−/− (filled columns) mice at day 10 of pregnancy were injected i.p. with 0.4 mg/kg LPS. Circulating chemokine concentrations were measured at 8 h postinjection by ELISA. Data are from nine WT and eight D6^−/− mice. (K–O) Chemokine levels in placenta. WT (open columns) and D6^−/− (filled columns) mice at day 10 of pregnancy were injected i.p. with 0.4 mg/kg LPS. Chemokine concentrations (expressed as nanograms of chemokine per milligram of total proteins of the lysates) were measured at 8 h postinjection by ELISA. Data are from nine WT and eight D6^−/− mice. Results are reported as mean ± SEM. (A, F, and K) CCL22. (B, G, and L) CCL2. (C, H, and M) CCL11. (D, I, and N) CCL3. (E, J, and O) CXCL2.

Fig. 5.

Placenta-infiltrating leukocytes in the LPS model of fetal loss. Leukocyte infiltration in placenta of WT (open symbols) and D6^−/− (filled symbols) mice at day 7 of pregnancy at the indicated time points after i.p. injection of 0.4 mg/kg LPS. The numbers of CD68⁺ macrophages (A), CD3⁺ lymphocytes (B), and Gr1⁺ neutrophils (C) infiltrating the placenta were evaluated on histological sections. Data are from at least three independent animals per group. Results are reported as mean ± SEM. ∗, P < 0.05 by Student's t test.