Stretch and inflammation-induced Pre-B cell colony-enhancing factor (PBEF/Visfatin) and Interleukin-8 in amniotic epithelial cells

Abstract

Preterm birth continues to be a growing problem in the USA. Although approximately half of preterm births are caused by intrauterine infection, uterine over-distension is also a cause. In this study we have compared the effects of static stretch, cyclic stretch/release and an inflammatory stimulus alone and in combination on the expression of Pre-B cell colony-enhancing factor (PBEF) and IL-8 in primary amniotic epithelial cells (AEC). We then sought to identify some of the mechanism(s) by which these cells respond to stretching stimuli. We show that cyclic stretch/release is a more robust stimulus for both PBEF and IL-8 than static stretch. Cyclic stretch/release increased both intracellular and secreted PBEF and a combination of both types of stretch was a more robust stimulus to PBEF that IL-8. However, when an inflammatory stimulus (IL-1beta) was added to either kind of stretch, the effect on IL-8 was much greater than that on PBEF. Thus, different kinds of stretch affect the expression of these two cytokines from AEC, but inflammation is a much stronger stimulus of IL-8 than PBEF, agreeing with its primary role as a chemokine. Although the AEC showed morphological signs of increased cellular stress during stretching, blocking reactive oxygen species (ROS) had little effect. However, blocking integrin binding to fibronectin significantly reduced the responses of both PBEF and IL-8 to cyclic stretch/release. The increased PBEF, both intracellularly and secreted, suggests that it functions both to increase the metabolism of the cells, at the same time as stimulating further the cytokine cascade leading to parturition.

Fig. 1

Effects of cyclic stretch/release on expression of PBEF and IL-8 in primary AEC (n=4 cells from different patients). (A) PBEF and (B) IL-8 gene expression during stimulation by cyclic stretch/release for 4 h. (C) PBEF and (D) IL-8 proteins secreted into medium after 24 h of cyclic stretch/release. Values are mean +/-SEM. *p<0.05, ** p<0.005 compared to control (no cyclic stretch/release).

Fig. 2

Effects of cyclic stretch/release on expression of intracellular PBEF protein. Immunofluorescence labeling of PBEF in AEC during 4 h of cyclic stretch/release stimulation. (A) T0 shows diffuse cytoplasmic labeling of PBEF, (B) at 15 mins there was increased intensity of PBEF labeling which continued during (C) 1 h and (D) 4 h of stimulation. (E) An example of a Western blot of PBEF with GAPDH as control using AEC lysates. Below, quantitation of Western data from n=4 (cells from different patients). Values are mean +/-SEM. *p<0.05 compared to control (no cyclic stretch/release).

Fig. 3

Effects of static stretch and cyclic stretch/release on PBEF and IL-8 gene expression in AEC (n=4) from different patients. (A) PBEF (B) IL-8. Values are mean +/-SEM. P<0.05 for dissimilar letters.

Fig. 4

The combined effect(s) of stretch and an inflammatory stimulus (IL-1β (1ng/ml)) on PBEF and IL-8 gene expression in AEC. (A) PBEF (B) IL-8. Values are mean +/- SEM. P<0.05 for dissimilar letters.

Fig. 5

The role of reactive oxygen species (ROS) in the responses of PBEF and IL-8 to stretching was shown by the specific blocking of ROS by NAC (30μM). In AEC the localization of stress fibers (white arrows) and filapodia (green arrows) in primary AEC during 4 h of static stretching. In WISH cells (n=6) static and cyclic stretch/release increased both PBEF (B) and IL-8 (C) gene expression. Values are mean +/-SEM.

Fig. 6

Integrins were activated in AEC by the cyclic stretch/release stimulus, shown by immunofluorescent localization of integrin β1 subunit (A-D). Clustering on the basal surface of the AEC (white arrows) can be seen after 15 mins of stimulation, maintained at both 1 h and 4 h of continued cyclic stretch/release. (E) Western blot of p-FAK with AEC lysates during 1 h of cyclic stretch/release, with GAPDH as control and quantitation (n=4) cells from different patients shown below. (F) Western blot of p-Paxillin of the same cell lysates shown during 1 h of cyclic stretch/release, with GAPDH as control and quantitation (n=4) shown below. Values are mean +/-SEM. *p<0.05 compared to no stretch control.

Fig. 7

The role of integrins in PBEF and IL-8 responses to stretching was shown by addition of a blocking peptide (RGD) to WISH cells during static and cyclic stretch/release (n=5). (A) PBEF (C) IL-8 gene expression with static stretch and (B) PBEF and (D) IL-8 with 2 h of cyclic stretch/release. An example experiment completed with primary isolated AEC from one patient showing gene expression for (E) PBEF and (F) IL-8. Values are mean +/-SEM. P<0.05 for dissimilar letters.