Monocyte adhesion to and transmigration through endothelium following cardiopulmonary bypass shearing is mediated by IL-8 signaling

Abstract

Introduction: The use of cardiopulmonary bypass (CPB) can induce sterile systemic inflammation that contributes to morbidity and mortality, especially in children. Patients have been found to have increased expression of cytokines and transmigration of leukocytes during and after CPB. Previous work has demonstrated that the supraphysiologic shear stresses existing during CPB are sufficient to induce proinflammatory behavior in non-adherent monocytes. The interactions between shear stimulated monocytes and vascular endothelial cells have not been well studied and have important translational implications. With these studies, we tested the hypothesis that non-physiological shear stress experienced by monocytes during CPB affects the integrity and function of the endothelial monolayer.

Methods: We have used an in vitro CPB model to study the interaction between THP-1 monocyte-like cells and human neonatal dermal microvascular endothelial cells (HNDMVECs). THP-1 cells were sheared in polyvinyl chloride (PVC) tubing at 2.1 Pa, twice of the physiological shear stress, for 2 h. ELISA, adhesion and transmigration assays, qPCR, and RNA silencing were used to assess the interactions between THP-1 cells and HNDMVECs were characterized after co-culture.

Results: We found that sheared THP-1 cells adhered to and transmigrated through the HNDMVEC monolayer more readily than static THP-1 controls. Sheared THP-1 cells disrupted the VE-cadherin and led to the reorganization of cytoskeletal F-actin of HNDMVECs. A higher level of IL-8 was detected in the sheared THP-1 and HNDMVEC co-culture medium compared to the static THP-1 and HNDMVEC medium. Further, treating HNDMVECs with IL-8 resulted in increased adherence of non-sheared THP-1 cells, and upregulation in HNDMVECs of vascular cell adhesion molecule 1 (VCAM-1) and intercellular adhesion molecule 1 (ICAM-1). Finally, inhibition of HNDMVECs CXCR2/IL-8 receptor with Reparixin and of IL-8 expression with siRNA blocked sheared THP-1 cell adhesion to the endothelial monolayer.

Conclusions: These results suggest that CPB-like sheared monocytes promote IL-8 production followed by increased endothelium permeability, and monocyte adhesion and transmigration. This study revealed a novel mechanism of post-CPB inflammation and will contribute to the development of targeted therapeutics to prevent and repair the damage to neonatal patients.

Keywords: IL-8; cardiopulmonary bypass; endothelial cells; monocytes; shear stress.

Figure 1

For cell adhesion assay, G-THP-1 cells were sheared in the in vitro CPB circuit for 2 h or cultured statically in a PVC flask, followed by co-culture with confluent HNDMVECs for 1 h. At the end of the co-culture, non-adherent G-THP-1 cells were rinsed, and adherent G-THP-1 cells were quantified. (A) Quantitative analysis of adhered G-THP-1 cells on the endothelial cell monolayer. (B) Adhesion of G-THP-1 cells incubated statically in a PVC flask. (C) Adhesion of G-THP-1 cells sheared in a CPB circuit. For transmigration assay, G-THP-1 cells were sheared in the in vitro CPB circuit for 2 h or cultured statically in a PVC flask. The G-THP-1 cells were then added to confluent HNDMVECs grown on the top chamber of the transwell insert. After an overnight incubation, the G-THP-1 cells transmigrated to the bottom chambers were quantified. (D) Quantitative analysis of transmigrated THP-1 cells through endothelial cell monolayer. (E) Transmigrated G-THP-1 cells incubated statically in a PVC flask. (F) Transmigration of G-THP-1 cells sheared in a CPB circuit. Scale bar = 100 μm. For additional images see Supplementary Figure S2. All experiments were run in triplicate. All quantitative data were expressed as mean ± standard deviation within groups. Pairwise comparisons between groups were conducted using the ANOVA test and Tukey's post-hoc test. Statistical significance is denoted by “*”. P values less than 0.05 are indicated by single symbols and P values less than 0.01 are indicated by double symbols.

Figure 2

VE-cadherin of HNDMVECs co-cultured with (A) sheared THP-1 cells, (B) THP-1 cells statically incubated in PVC flask, and (C) No THP-1 treatment, visualized by immunofluorescent staining. For additional images see Supplementary Figure S3. Red = VE-Cadherin, Green = THP-1 Cells, Blue = DAPI. Scale bar = 100 μm. (D) Quantification of VE cadherin staining. G-THP-1 cells were sheared in the in vitro CPB circuit for 2 h or cultured statically in a PVC flask, followed by co-culture with confluent HNDMVECs for 6 h. After the co-culture, the cells were fixed and stained with VE-cadherin antibodies. All experiments were run in triplicate. All quantitative data were expressed as mean ± standard deviation within groups. Pairwise comparisons between groups were conducted using the ANOVA test and Tukey's post-hoc test. Statistical significance is denoted by “*”. P values less than 0.05 are indicated by single symbols and P values less than 0.01 are indicated by double symbols.

Figure 3

F-actin of HNDMVECs co-cultured with (A) sheared THP-1 cells, (B) THP-1 cells statically incubated in PVC flask, and (C) No THP-1 treatment, stained by rhodamine-phalloidin. For additional images see Supplementary Figure S4. Red = F-actin, Green = THP-1 Cells, Blue = DAPI. Scale bar = 100 μm. (D) Quantification of intercellular gap area of endothelial cells. All experiments were run in triplicate. All quantitative data were expressed as mean ± standard deviation within groups. Pairwise comparisons between groups were conducted using the ANOVA test and Tukey's post-hoc test. Statistical significance is denoted by “*”. P values less than 0.05 are indicated by single symbols and P values less than 0.01 are indicated by double symbols.

Figure 4

Cytokine levels of (A) IL-8, (B) IL-6, and (C) IL-1β in the co-culture media of sheared or static THP-1 cells and HNDMVECs measured at 0.5, 1, 3, and 6 h. All experiments were run in triplicate. All quantitative data were expressed as mean ± standard deviation within groups. Pairwise comparisons between groups were conducted using the ANOVA test and Tukey's post-hoc test. Statistical significance is denoted by “*”. P values less than 0.05 are indicated by single symbols and P values less than 0.01 are indicated by double symbols.

Figure 5

(A) quantitative analysis of sheared G-THP-1 cells adhering to (B) untreated HNDMVECs, (C) HNDMVECs pretreated with 5nM reparixin for 30 min, and (D) HNDMVECs pretreated with PBS. Scale bar = 100 μm. For additional images see Supplementary Figure S6. All experiments were run in triplicate. All quantitative data were expressed as mean ± standard deviation within groups. Pairwise comparisons between groups were conducted using the ANOVA test and Tukey's post-hoc test. Statistical significance is denoted by “*”. P values less than 0.05 are indicated by single symbols and P values less than 0.01 are indicated by double symbols.

Figure 6

(A) quantitative analysis of statically incubated G-THP-1 cells adhering to (B) untreated HNDMVECs, (C) HNDMVECs pretreated with 50 ng/ml IL-8 for 1 h, and (D) HNDMVECs pretreated with 200 ng/ml IL-8 for 1 h. For additional images see Supplementary Figure S7. Scale bar = 100 μm. All experiments were run in triplicate. All quantitative data were expressed as mean ± standard deviation within groups. Pairwise comparisons between groups were conducted using the ANOVA test and Tukey's post-hoc test. Statistical significance is denoted by “*”. P values less than 0.05 are indicated by single symbols and P values less than 0.01 are indicated by double symbols.

Figure 7

(A) quantitative analysis of (B) statically incubated G-THP-1 cells adhering to HNDMVECs, and sheared G-THP-1 cells adhering to (C) HNDMVECs, (D) HNDMVECs treated with IL-8 siRNA, and (E) HNDMVECs treated with negative control siRNA. For additional images see Supplementary Figure S9. Scale bar = 100 μm. All experiments were run in triplicate. All quantitative data were expressed as mean ± standard deviation within groups. Pairwise comparisons between groups were conducted using ANOVA test and Tukey's post-hoc test. Statistical significance is denoted by “*”. P values less than 0.05 are indicated by single symbols and P values less than 0.01 are indicated by double symbols.

Figure 8

Expression of e-selectin, ICAM1, and VCAM1 in HNDMVECs treated with IL-8, TNF-α, and control measured by RT-qPCR. All experiments were run in triplicate. All quantitative data were expressed as mean ± standard deviation within groups. Pairwise comparisons between groups were conducted using ANOVA test and Tukey's post-hoc test. Statistical significance is denoted by “*”. P values less than 0.05 are indicated by single symbol and P values less than 0.01 are indicated by double symbols.

Figure 9

Cartoon describing a proposed mechanism of shear stress activation THP1 effect on endothelial cells.

Figure 10

Cartoon representing a working model of the shear stress and IL-8 signaling CPB-induced inflammatory response.