Stretch-induced human myometrial cytokines enhance immune cell recruitment via endothelial activation

Abstract

Spontaneous term labour is associated with amplified inflammatory events in the myometrium including cytokine production and leukocyte infiltration; however, potential mechanisms regulating such events are not fully understood. We hypothesized that mechanical stretch of the uterine wall by the growing fetus facilitates peripheral leukocyte extravasation into the term myometrium through the release of various cytokines by uterine myocytes. Human myometrial cells (hTERT-HM) were subjected to static mechanical stretch; stretch-conditioned media was collected and analysed using 48-plex Luminex assay and ELISA. Effect of stretch-conditioned media on cell adhesion molecule expression of human uterine microvascular endothelial cells (UtMVEC-Myo) was detected by quantitative polymerase chain reaction (qPCR) and flow cytometry; functional assays testing leukocyte-endothelial interactions: adhesion of leukocytes to endothelial cells and transendothelial migration of calcein-labelled primary human neutrophils as well as migration of THP-1 monocytic cells were assessed by fluorometry. The current in vitro study demonstrated that mechanical stretch (i) directly induces secretion of multiple cytokines and chemokines by hTERT-HM cells (IL-6, CXCL8, CXCL1, migration inhibitory factor (MIF), VEGF, G-CSF, IL-12p70, bFGF and platelet-derived growth factor subunit B (PDGF-bb), P<0.05); stretch-induced cytokines (ii) enhance leukocyte adhesion to the endothelium of the surrounding uterine microvasculature by (iii) inducing the expression of endothelial cell adhesion molecules and (iv) directing the transendothelial migration of peripheral leukocytes. (vi) Chemokine-neutralizing antibodies and broad-spectrum chemokine inhibitor block leukocyte migration. Our data provide a proof of mechanical regulation for leukocyte recruitment from the uterine blood vessels to the myometrium, suggesting a putative mechanism for the leukocyte infiltrate into the uterus during labour and postpartum involution.

Figure 1

Pro-inflammatory cytokines are up-regulated by static stretch in hTERT-HM cells. Cytokines released by hTERT-HM cells in culture media upon 24 h of 25% static stretch (n=7). White bars represent cytokines detected in the non-stretched conditioned media (NS-CM), whereas black bars represent cytokines detected in the stretched conditioned media (S-CM). (a) Absolute concentration values are presented as mean±s.e.m. on a logarithmic scale. (b) Increase in cytokine levels secreted in S-CM is illustrated as fold change relative to NS-CM levels (the dotted line in the bottom designates 1). Significance was set at *P<0.05 comparing each S-CM group to its corresponding NS-CM group.

Figure 2

Stretch-conditioned media activates human microvascular endothelial cells through enhanced cell adhesion molecule expression and vascular permeability. (a) Confluent endothelial cells (hUtMVEC-Myo) were stimulated with NS-CM or S-CM for 4 h and the expression of E-selectin, ICAM-1, VCAM-1 and PECAM-1 genes were analysed by RT-PCR (unpaired t-test, n=3). Expression of cell adhesion molecules on S-CM-treated hUtMVEC-Myo is presented as fold change relative to corresponding expression of NS-CM-treated cells (the dotted line in the bottom designates 1). (b) Representative FACS histogram overlay comparing surface protein expression of ICAM-1 on hUtMVEC-Myo (n=6) after incubation with serum-free media (SF-DMEM, blue), NS-CM (green) and S-CM (red). FMO controls (grey) were used to determine the boundary of individual gates. (c) Gated values (percentage of positive cells) and (d) MFI values of cell adhesion molecule proteins expressed on hUtMVEC-Myo treated with NS-CM (white bars) and S-CM (black bars). Data was analysed by unpaired t-test (n=6). (e) Confluent hUtMVEC-Myo grown on 3.0-µm inserts were stimulated with NS-CM (white bars), S-CM (black bars) or endothelial growth media (control, striped bars) for 4 h and the leakage of FITC-dextran was analysed by fluorescence. Bar graphs represent leakage percentage of each treatment relative to the maximum leakage (100%) detected in the absence of endothelial cells. Data was analysed by one-way ANOVA with Tukey's post-test (n=4). For a, c, d and e: *P<0.05, **P<0.01, ***P<0.001 compared to NS-CM group; ^#P<0.05 compared to control. All values are presented as mean±s.e.m. FMO, fluorescence minus one; MFI, median fluorescence intensity.

Figure 3

Multiple cytokines released from human myometrial smooth muscle cells activate peripheral leukocytes. Representative FACS histogram overlay plots of CD11b and CD44 protein expression on surface of peripheral granulocytes (Grans) and monocytes (Monos) following stimulation with SF-DMEM (blue, negative control), NS-CM (green) and S-CM (red). Fluorescence minus one controls (‘FMO', grey) were used to determine the boundary of individual gates. Basal expression levels of both leukocyte protein markers prior to activation (at time ‘0') are represented by ‘BASAL' (magenta). (a) CD11b expression was examined after 1 h of stimulation (representative of five separate experiments). (b) CD44 expression was examined after 6 h of stimulation (representative of three experiments).

Figure 4

Stretch-conditioned media induces adhesion of peripheral human leukocytes to endothelial cells and transendothelial migration. (a) Confluent hUtMVEC-Myo cells grown on cell culture plate were stimulated for 3 h with conditioned media. Calcein-labelled primary human neutrophils adhered to stimulated endothelial monolayer (NS-CM, white bar; S-CM, black bar). Bar graphs represent fold changes of adherent neutrophil number relative to serum-free media control. Data were analysed by unpaired t-test (n=4). (b–d) Peripheral human neutrophils pre-treated with either anti-CXCR1, anti-CXCR2 or both blocking antibodies (10 µg/ml) were labelled with calcein-AM and incubated with (a) conditioned media-stimulated (NS-CM or S-CM), (c) CXCL8-stimulated or (d) CXCL1-stimulated endothelial cells grown on membrane inserts (grey bars). Primary neutrophil TEM (percentage of transmigrated cells) was measured. Serum-free media (SF-DMEM) served as controls in c and d (striped bars). Graphs are representative of two independent experiments with three replicates in each treatment group. (e) In addition, BSCI (2 nM, checkered bar) was added to the S-CM and TEM was measured (n=4). (f) Monocytic cell line THP-1 migration towards NS-CM/S-CM and BSCI-treated S-CM was also examined (n=5). (e and f) Results are shown as fold change of migrated neutrophils or THP-1 cells through membrane inserts relative to SF-DMEM control. (b–f) Values are presented as mean±s.e.m. Data was analysed by one-way ANOVA followed by Tukey's multiple comparison test. *P<0.05, **P<0.01, ***P<0.001, compared to NS-CM group or control. BSCI, broad-spectrum chemokine inhibitor; TEM, transendothelial migration.

Figure 5

Putative model of human labour. Our data suggested that (1) mechanical stretch of the myometrium by the growing fetus induces the secretion of multiple cytokines and chemokines near term. We also demonstrated that stretch-induced cytokines (2) activate peripheral leukocytes and (3) enhance the expression of endothelial CAMs. This resulted in (4) enhanced adhesion of immune cells to myometrial vascular endothelial cells, promoting (5) leukocyte transendothelial migration into the uterine muscle. Within the muscle, immune cells differentiate, producing more cytokines/chemokines, and contribute to the amplification of inflammatory signal that ultimately leads to the onset of labour and facilitates the process of postpartum involution. CAM, cell adhesion molecule.