Abstract
The epidermal growth factor receptor (EGFR) and its family of ligands may be associated with the progression of atherosclerosis by mediating the proliferation and migration of endothelial cells and vascular smooth muscle cells. However there is some evidence that EGFR is expressed by cells of the monocytic lineage. This study was undertaken to investigate the expression of the EGFR on monocytes and macrophages and explore its potential function. The expression of the EGFR was studied using the monoclonal antibody ICR62. Flow cytometry revealed that the monocytic THP-1 cell line expressed EGFR on its surface and specific EGFR reactivity was also observed by western blotting analysis. Whilst PMA differentiated THP-1 cells failed to show cell surface expression of the receptor, immunocytochemical analysis revealed the receptor was localized to the nucleus. Human peripheral blood-derived monocytes and monocyte-derived macrophages showed minimal cell surface expression of EGFR. EGFR gene transcription in monocytic THP-1 cells was induced 2.7 fold following treatment with 50nM HB-EGF for 12 hours and this was statistically significant (P>0.01). Differentiated THP-1 cells showed a 1.5 fold increase in gene expression following treatment with 50nM TGFa for 12 hours; this increase was also highly significant (P<0.01). Gene expression was also significantly increased in both monocytic and differentiated THP-1 cells following 12 hours treatment with 100U/ml IFNy [3.7 fold (P<0.01) and 1.8 fold (P<0.01) respectively] and 2.5ng/ml IL-lp [3.6 fold (P<0.01) and 1.7 fold (P<0.05) respectively)]. Newly isolated human peripheral blood-derived monocytes displayed very low EGFR mRNA expression. However, during maturation in vitro, gene expression peaked following 48 hours in culture and returned to baseline after 96 hours. Exposure of THP-1 cells to HB-EGF (0.1 nM) resulted in a 3 fold increase (P<0.01) in maximum chemotactic activity. This response was found to be greater than the 2 fold increase seen with the well established monocyte chemokine MCP-1 at 1nM (P<0.05). Peripheral blood-derived monocytes had a statistically significant (P<0.01) 2.4 fold increase in their chemotactic response compared to unstimulated control cells when exposed to 10nM HB-EGF. This was equivalent to the response seen for 0.1nM MCP-1 with these same cells. Interestingly, as the monocyte matured into an 8 day old macrophage-like cell the chemotactic response to 0.1nM MCP-1 decreased, whereas peak chemotactic activity to HB-EGF occurred at lower concentrations. Incubation of peripheral blood-derived monocytes with the inhibitory monoclonal antibody ICR62 significantly (P<0.05) reduced chemotactic activity towards HB-EGF (0.1nM) in a dose dependent manner. Immunohistochemical analysis of human arterial sections revealed EGFR expression within atherosclerotic lesions. Further analysis showed that EGFR expression colocalised with macrophages. It therefore appears that the EGFR is expressed by the THP-1 cell line and human peripheral blood-derived monocytes and may play a role in the pathogenesis of human atherosclerosis.