Interleukin-1alpha released from epithelial cells after adenovirus type 37 infection activates intercellular adhesion molecule 1 expression on human vascular endothelial cells

Abstract

A key event in virus-induced inflammation (leukocyte extravasation through the endothelium) is the local activation of endothelial cells, as indicated by the expression of adhesion molecules such as intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1), and E-selectin. In order to identify triggers of inflammation in adenovirus infection, we inoculated respiratory and ocular epithelial cells with adenovirus type 37 (Ad37), a human pathogen associated with keratoconjunctivitis as well as urogenital and respiratory infections. Fluids from virus-infected epithelial cells activated ICAM-1 (and to a lesser extent, VCAM-1) expression on cultured human umbilical vein endothelial cells. Blocking studies with anticytokine antibodies implicated interleukin-1alpha (IL-1alpha) as the epithelial cell-derived factor which activated endothelial cell ICAM-1 expression. The results thus identify epithelial cell-derived IL-1alpha as a potentially important activator of endothelial cells in Ad37-induced inflammation.

FIG. 1.

ICAM-1 expression on vascular endothelial cells is upregulated after treatment (18 h) with culture supernatants from Ad-infected A549 cells or Ad-infected human corneal epithelial cells. (A) Endothelial ICAM-1 expression after 18-h exposure to culture fluids from Ad-infected A549 cells, mock-infected A549 cells, and medium alone (negative control; NC). (B) Endothelial ICAM-1 expression after 18-h exposure to culture fluids from Ad-infected human corneal epithelial cells, mock-infected A549 cells, and medium alone (negative control; NC). PC, positive control (vascular endothelial cells treated for 18 h with TNF-α [10 U/ml]). Data are from three to nine separate experiments with triplicate samples and are represented as the means ± standard deviations of the means. Significant differences from the mock sample are indicated by * (P < 0.05) and ** (P < 0.01).

FIG. 2.

Effects of culture supernatants from Ad-infected A549 cells on endothelial cell expression of VCAM-1 and E-selectin. (A) Endothelial VCAM-1 expression after 18-h exposure to culture fluids from Ad-infected A549 cells, mock-infected A549 cells, and medium alone (negative control; NC). (B) Endothelial E-selectin expression after 3-h exposure to culture fluids from Ad-infected A549 cells, mock-infected A549 cells, and medium alone (negative control; NC). PC, positive control (vascular endothelial cells treated for 18 h with TNF-α [10 U/ml]). Data are from three separate experiments with triplicate samples and are represented as the means ± standard deviations of the means. Significant differences from the mock sample are indicated by * (P < 0.05).

FIG. 3.

ICAM-1 expression on vascular endothelial cells is activated by recombinant cytokines. (A) ICAM-1 expression on endothelial cells incubated for 18 h with IL-1α (4 pg/ml), IL-1β (100 pg/ml), TNF-α (100 U/ml), IL-6 (1 μg/ml), and IL-8 (1 μg/ml). (B) ICAM-1 expression on endothelial cells incubated for 18 h with IL-1α in the presence or absence of anti-IL-1α antibody at the indicated concentration. (C) ICAM-1 expression on endothelial cells incubated for 18 h with IL-1β in the presence or absence of anti-IL-1β antibody at the indicated concentration. (D) ICAM-1 expression on endothelial cells incubated for 18 h with TNF-α in the presence or absence of anti-TNF-α antibody at the indicated concentration. NC, negative control (medium alone); PC, positive control (vascular endothelial cells treated for 18 h with TNF-α [10 U/ml]). Data are from three to six separate experiments with triplicate samples and are represented as the means ± standard deviations of the means. Significant differences between antibody-treated and untreated samples are indicated by * (P < 0.05) and ** (P < 0.01).

FIG. 4.

Identification of endothelial ICAM-1-activating factor released from Ad-infected epithelial cells as IL-1α. ICAM-1 expression on vascular endothelial cells after stimulation with culture fluids from Ad-infected (72 h) A549 cells (A), Ad-infected (24 h) corneal epithelial cells (B), or Ad-infected (72 h) corneal epithelial cells (C) is blocked by anti-IL-1α antibody (20 μg/ml) but not by other anticytokine antibodies. Inf, infected cells; Sup, supernatant; NC, negative control (medium alone); PC, positive control (vascular endothelial cells treated for 18 h with TNF-α [10 U/ml]). Data are from three to five separate experiments with triplicate samples and are represented as the means ± standard deviations of the means. Significant differences between antibody-treated and untreated samples are indicated by ** (P < 0.01).