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. 2004 Mar;78(6):2831-40.
doi: 10.1128/jvi.78.6.2831-2840.2004.

Human cytomegalovirus interleukin-10 downregulates metalloproteinase activity and impairs endothelial cell migration and placental cytotrophoblast invasiveness in vitro

Takako Yamamoto-Tabata  1 , Susan McDonagh, Hsin-Ti Chang, Susan Fisher, Lenore Pereira
Affiliations

Human cytomegalovirus interleukin-10 downregulates metalloproteinase activity and impairs endothelial cell migration and placental cytotrophoblast invasiveness in vitro

Takako Yamamoto-Tabata et al. J Virol. 2004 Mar.

Abstract

At the uterine-placental interface, fetal cytotrophoblasts invade the decidua, breach maternal blood vessels, and form heterotypic contacts with uterine microvascular endothelial cells. In early gestation, differentiating- invading cytotrophoblasts produce high levels of matrix metalloproteinase 9 (MMP-9), which degrades the extracellular matrix and increases the invasion depth. By midgestation, when invasion is complete, MMP levels are reduced. Cytotrophoblasts also produce human interleukin-10 (hIL-10), a pleiotropic cytokine that modulates immune responses, helping to protect the fetal hemiallograft from rejection. Human cytomegalovirus (CMV) is often detected at the uterine-placental interface. CMV infection impairs cytotrophoblast differentiation and invasion, altering the expression of the cell adhesion and immune molecules. Here we report that infection with a clinical CMV strain, VR1814, but not a laboratory strain, AD169, downregulates MMP activity in uterine microvascular endothelial cells and differentiating-invading cytotrophoblasts. Infected cytotrophoblasts expressed CMV IL-10 (cmvIL-10) mRNA and secreted the viral cytokine, which upregulated hIL-10. Functional analyses showed that cmvIL-10 treatment impaired migration in endothelial cell wounding assays and cytotrophoblast invasion of Matrigel in vitro. Comparable changes occurred in cells that were exposed to recombinant hIL-10 or cmvIL-10. Our results show that cmvIL-10 decreases MMP activity and dysregulates the cell-cell and/or cell-matrix interactions of infected cytotrophoblasts and endothelial cells. Reduced MMP activity early in placental development could impair cytotrophoblast remodeling of the uterine vasculature and eventually restrict fetal growth in affected pregnancies.

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Figures

FIG. 1.
FIG. 1.
Anatomy of the maternal-fetal interface, where the fetus-derived placenta attaches to the mother's uterus. The basic structural unit of the placenta is the chorionic villus, composed of a stromal core with blood vessels, surrounded by a basement membrane, and overlaid by cytotrophoblast stem cells. As part of their differentiation pathway, stem cells detach from the basement membrane and adopt one of two lineage fates. They fuse to form the syncytiotrophoblast, which covers floating villi, or they join a column of extravillus cytotrophoblasts that invade the uterine stroma. The syncytiotrophoblast mediates nutrient and gas exchange across the maternal-fetal interface. The anchoring villi (AV) establish physical connections between the mother and fetus through the attachment of cytotrophoblast columns. The floating villi (FV), bathed by maternal blood, contain the fetal capillaries (zone I). Cytotrophoblasts in the AV attach the placenta to the uterine wall (zone II). Cytotrophoblasts then invade the decidua up to the first third of the myometrium (zone III), anchoring the placenta to the uterus and gaining access to the maternal circulation. Sites proposed as routes of CMV infection in utero are numbered 1 to 4.
FIG. 2.
FIG. 2.
Downregulation of MMP-2 in CMV-infected human fibroblasts. CM and lysates from AD169- and VR1814-infected fibroblasts and uninfected fibroblasts were analyzed 4 days after infection by gelatin zymography (A and B) and immunoblotting (C and D). CMV reduced MMP-2 expression (both the 72-kDa proenzyme and the 66-kDa activated form) in CM (A and C) and cell lysates (B and D). MMP-2 activity was quantified in VR1814-infected fibroblast CM and cell lysates as a percentage of the control (E). Values are the means of four experiments and were highly reproducible. Asterisks indicate a significant difference between control and infected cells (P < 0.05; Fisher exact test). Error bars show SDs.
FIG. 3.
FIG. 3.
Downregulation of MMP-2 and MMP-9 activity in VR1814-infected UtMVEC. (A and B) CM and lysates were analyzed by gelatin zymography after 1 and 7 days (uninfected control cells) and 7 days after the infection of experimental cells. (C) MMP-9 and MMP-2 activity in CM and cell lysates was quantified as a percentage of the control. Values are the means of four experiments. Asterisks indicate a significant difference between control and infected cells (P < 0.05). Error bars show SDs.
FIG. 4.
FIG. 4.
Expression of cmvIL-10 in VR1814-infected cells. (A) RT-PCR analysis of total RNA isolated 4 days after infection. HF, human fibroblasts. (B) Immunoblot analysis of cmvIL-10 protein complexes immunoprecipitated from infected cells 5, 7, and 12 days after infection (dpi).
FIG. 5.
FIG. 5.
CMV-infected endothelial cells secrete a soluble product that diminishes MMP-2 activity. Uninfected HUVEC were cultured for 6 days with CM from VR1814-infected UtMVEC (inf) or control cells (cont). CM and cell lysates were analyzed by gelatin zymography (A and B) and immunoblotting (C and D). MMP-2 activity was quantified in CM and lysates as a percentage of the control (E). Values are the means of two experiments. Asterisk indicates a significant difference between control and infected cells (P < 0.05). Error bars show SDs. UtMVEC that were infected or treated with purified recombinant cmvIL-10 or hIL-10 downregulated MMP-2 activity in CM (F) and cell lysates (G).
FIG. 6.
FIG. 6.
Reduced MMP-9 activity in differentiating-invading cytotrophoblasts infected with VR1814 or treated with cmvIL-10. Cytotrophoblasts were untreated (CTB), infected, treated with CM, or treated with recombinant hIL-10 (h) or cmvIL-10 (cmv). Matrigel-coated wells without cells served as a control (Matrigel). CM and cell lysates were then analyzed for MMP-9 activity by gelatin zymography (A and C) and immunoblotting (B and D). CMV infection, hIL-10 treatment, and cmvIL-10 (100 ng/ml) treatment all decreased MMP-9 (both the 92-kDa proenzyme and the 86-kDa activated form) in CM (A and B) and cell lysates (C and D). Cytotrophoblasts were infected or treated with hIL-10 or cmvIL-10 for 4 days, and the CM (E) and lysates (F) were analyzed for MMP-9 activity, which was quantified as a percentage of the control. Values are the means of five experiments. Asterisks indicate a significant difference between control and infected or IL-10-treated cells (P < 0.05). Error bars show SDs.
FIG. 7.
FIG. 7.
Upregulation of hIL-10 expression in differentiating-invading cytotrophoblasts after VR1814 infection and cmvIL-10 treatment. CM from VR1814- and mock-infected control or cmvIL-10-treated cytotrophoblasts was collected on day 1 and cultured for 24 to 36 h. hIL-10 was then quantified by ELISA. Results are expressed as percentages of the control. CM from untreated mock-infected cells served as a control. Values are the means of three experiments. Asterisks indicate a significant difference between control and infected or treated cells (P < 0.05). Error bars show SDs.
FIG. 8.
FIG. 8.
Impaired cell motility in functional assays of endothelial cell wound healing and cytotrophoblast invasion of Matrigel in vitro. Human fibroblasts (A) and HUVEC (B) were infected with VR1814 and treated with hIL-10 or cmvIL-10 (100 ng/ml). The horizontal lines indicate the original widths of the wounded cell sheets. (C) Differentiating-invading cytotrophoblasts from first-trimester placentas were plated on Matrigel-coated filters and then were treated with cmvIL-10 or hIL-10, fixed at 48 h, and stained with a cytokeratin-specific antibody. Cells that reached the filter underside were counted. Values (percentages of the control) are expressed as the means ± SDs of three experiments. Asterisks indicate a significant difference between control and IL-10-treated cells (P < 0.05).
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