Selective regulation of MAP kinases and chemokine expression after ligation of ICAM-1 on human airway epithelial cells

Abstract

Background: Intercellular adhesion molecule 1 (ICAM-1) is an immunoglobulin-like cell adhesion molecule expressed on the surface of multiple cell types, including airway epithelial cells. It has been documented that cross-linking ICAM-1 on the surface of leukocytes results in changes in cellular function through outside-inside signaling; however, the effect of cross-linking ICAM-1 on the surface of airway epithelial cells is currently unknown. The objective of this study was to investigate whether or not cross-linking ICAM-1 on the surface of airway epithelial cells phosphorylated MAP kinases or stimulated chemokine expression and secretion.

Methods: The human lung adenocarcinoma (A549) cells and primary cultures of normal human bronchial epithelial (NHBE) cells were used in these studies. To increase ICAM-1 surface expression, cultures were stimulated with TNFalpha to enhance ICAM-1 surface expression. Following ICAM-1 upregulation, ICAM-1 was ligated with a murine anti-human ICAM-1 antibody and subsequently cross-linked with a secondary antibody (anti-mouse IgG(ab')2) in the presence or absence of the MAP kinase inhibitors. Following treatments, cultures were assessed for MAPK activation and chemokine gene expression and secretion. Control cultures were treated with murine IgG1 antibody or murine IgG1 antibody and anti-mouse IgG(ab')2 to illustrate specificity. Data were analyzed for significance using a one-way analysis of variance (ANOVA) with Bonferroni post-test correction for multiple comparisons, and relative gene expression was analyzed using the 2-DeltaDeltaCT method.

Results: ICAM-1 cross-linking selectively phosphorylated both ERK and JNK MAP kinases as detected by western blot analysis. In addition, cross-linking resulted in differential regulation of chemokine expression. Specifically, IL-8 mRNA and protein secretion was not altered by ICAM-1 cross-linking, in contrast, RANTES mRNA and protein secretion was induced in both epithelial cultures. These events were specifically inhibited by the ERK inhibitor PD98059. Data indicates that ICAM-1 cross-linking stimulates a synergistic increase in TNFalpha-mediated RANTES production involving activation of ERK in airway epithelial cells.

Conclusion: Results demonstrate that cytokine induced ICAM-1 on the surface of airway epithelial cells induce outside-inside signaling through cross-linking ICAM-1, selectively altering intracellular pathways and cytokine production. These results suggest that ICAM-1 cross-linking can contribute to inflammation in the lung via production of the chemokine RANTES.

Figure 1

Cross-linking of ICAM-1 induces phosphorylation of MAP kinases in airway epithelial cells. (Figure A. A549 cells) (Figure B. NHBE cells). Lane 1 represents cultures that were cross-linked with control IgG1 mAb and anti-mouse IgG F(ab')₂ for 10 min. Lanes 2–5 represent cultures that were cross-linked with anti-ICAM-1 mAb and anti-mouse IgG F(ab')₂ for 5 min (lane 2), 10 min (lane 3), 15 min (lane 4), and 30 min (lane 5). Lane 6 represents cultures that were exposed with anti-ICAM-1 mAb alone for 10 min. Lane 7 represents cultures that were exposed with anti-mouse IgG F(ab')₂ mAb alone for 10 min. Lane 8 represents cultures that were exposed to anti-VCAM-1 mAb and anti-mouse IgG F(ab')₂ for 10 min. Fold increase in amounts of phosphorylated MAP kinase proteins as indicated above are expressed as the mean ± SD in three different experiments.

Figure 2

Selective induction of chemokine mRNA by airway epithelial cultures cross-linked with ICAM-1. (Figure A. A549 cells) (Figure B. NHBE cells). Cultures were incubated with control IgG1 mAb and anti-mouse IgG F(ab')₂ (white columns) or anti-ICAM-1 mAb and anti-mouse IgG F(ab')₂ (black columns) for the indicated times before RNA was isolated for real time RT-PCR analysis of both RANTES and IL-8 message (40 PCR cycles). Messenger RNA of the 18s ribosomal gene was amplified under the same conditions and was used as the internal control. (n = 6; * = significantly different from control; p < 0.05).

Figure 3

Cross-linking of ICAM-1 induces selective chemokine secretion from airway epithelial cells. (Figure A. A549 cells) (Figure B. NHBE cells). Cultures were incubated with control IgG1 mAb and anti-mouse IgG F(ab')₂ or anti-ICAM-1 mAb and anti-mouse IgG F(ab')₂ for the indicated times. Both RANTES and IL-8 was detected in supernatants by ELISA.(n = 6; * = significantly different from control; p < 0.05).

Figure 4

Inhibition of ICAM-1 cross-linking induced phosphorylation of MAP kinases in airway epithelial cells (Figure A. A549 cells) (Figure B. NHBE cells). Cultures were pre-incubated in the presence of increasing concentrations of MAP kinase inhibitors. Cultures were then cross-linked for 10 minutes to assess ERK phosphorylation and 30 minutes to assess JNK phosphorylation. Lane 0 represents cultures that were cross-linked with control IgG1 mAb and anti-mouse IgG F(ab')₂ for 10 minutes for ERK phosphorylation and 30 minutes for JNK phosphorylation. Lane C represents a negative protein control prepared from cells incubated in media alone. Lane T represents cultures that were stimulated with 10 mg/ml TNF for 10 min to serve as a positive control. Cultures that were not cross-liked and pre-incubated with either MAP kinase inhibitor alone had no effect on MAP Kinase phosphorylation. Fold increase in amounts of phosphorylated ERK and JNK proteins are expressed as the mean ± SD in three different experiments.

Figure 5

Selective inhibition of ICAM-1 cross-linking induced RANTES mRNA in airway epithelial cultures by MAP kinase inhibitors (Figure A. A549 cells) (Figure B. NHBE cells). Cultures were pre-incubated in the presence of increasing concentrations of MAP kinase inhibitors and subsequently cross-linked for two hours with anti-ICAM-1 mAb and anti-mouse IgG F(ab')₂ before RNA was isolated for RT-PCR analysis of RANTES message (40 PCR cycles). Messenger RNA of the 18s ribosomal gene was amplified under the same conditions and was used as the internal control. Lane 0 represents cultures that were cross-linked with control IgG1 mAb and anti-mouse IgG F(ab')₂ for 2 hours. Cultures pre-incubated with MAP kinase inhibitors alone had no effect on RANTES or IL-8 mRNA. (n = 6; * = significantly different from control; † = significantly different from 0.5 μM PD98059; p < 0.05).

Figure 6

Inhibition of ICAM-1 cross-linking induced RANTES secretion from airway epithelial cultures by PD98059 (Figure A. A549 cells) (Figure B. NHBE cells). Epithelial cultures were pre-incubated for 30 minutes in the presence of increasing concentrations of MAP kinase inhibitors. Cultures were then incubated with anti-ICAM-1 mAb and anti-mouse IgG F(ab')₂ for 6 hours. Cultures pre-incubated with MAP kinase inhibitors alone had no effect on RANTES secretion. (Results represent means ± SD of triplicate measurements). (n = 6; * = significantly different from control; $ = significantly different from 0.0 μM PD98059; † = significantly different from 0.5 μM PD98059; p < 0.05).