Porphyromonas gingivalis-Induced MIF Regulates Intercellular Adhesion Molecule-1 Expression in EA.hy926 Cells and Monocyte-Endothelial Cell Adhesion Through the Receptors CD74 and CXCR4

Abstract

Porphyromonas gingivalis (P. gingivalis) is an important pathogen that contributes to periodontal disease and causes infections that promote the progression of atherosclerosis. Our previous studies showed that macrophage migration inhibitory factor (MIF) facilitates monocyte adhesion to endothelial cells by regulating the expression of intercellular adhesion molecule-1 (ICAM-1) in P. gingivalis-infected endothelial cells. However, the detailed pathological role of MIF has yet to be elucidated in this context. To explore the functional receptor(s) of MIF that underlie its participation in the pathogenesis of atherosclerosis, we investigated the expression of the chemokine receptors CD74 and CXCR4 in endothelial cells, both of which were shown to be involved in the adhesion of monocytes to endothelial cells pretreated with P. gingivalis. Furthermore, the formation of a MIF, CD74, and CXCR4 ligand-receptor complex was revealed by our immunofluorescence staining and coimmunoprecipitation results. By interacting with the CD74/CXCR4 receptor complex, MIF may act as a crucial regulator of monocyte-endothelial cell adhesion and promote the atherosclerotic plaque formation induced by P. gingivalis.

Keywords: CD74, CXCR4; Porphyromonas gingivalis; atherosclerosis; intercellular cell adhesion molecule-1; macrophage migration inhibitory factor.

Fig. 1

P. gingivalis infection induced the protein level of CXCR4 but not CD74 in EA.hy926 cells. P. gingivalis infected EA.hy926 cells for 24 h at MOI = 100, then the expression of CD74 and CXCR4 was detected by Western blot. Cells cultured without P. gingivalis were used as a control. a Western blot analysis of CD74 and CXCR4 in endothelial cells. b Quantitative analysis of the Western blot. Data were presented compared to the GAPDH reference. *P < 0.01.

Fig. 2

P. gingivalis induction of ICAM-1 and ICAM-1 mRNA expression is partially dependent on CD74 and CXCR4. EA.hy926 cells were infected with P. gingivalis at MOI = 100 for 24 h after the addition of C-16 or AMD3100, then the level of ICAM-1 and ICAM-1 mRNA was determined by Western blot and qRT-PCR. EA.hy926 cells cultured in medium only were used as a negative control. a Western blot analysis of ICAM-1.b Quantitative analysis of Western blot. c Quantitative real-time PCR analysis of ICAM-1 mRNA. *P < 0.01.

Fig. 3

Enhanced THP-1 cells adhesion to P. gingivalis-infected EA.hy926 cells is partially dependent on CD 74 and CXCR4. The EA.hy926 cells were pre-incubated with C-16 (5 μg/mL) or AMD3100 (20 nM) for 1 h then infected with P. gingivalis for 24 h (MOI = 100). THP-1 cells labeled with Calcein-AM (5 μM) were co-cultured with EA.hy926 cells for additional 1 h before the adhesion assay. The control group was EA.hy926 cells pre-treated with culture medium only.a Calcein-AM labeled THP-1 cells adhered to EA.hy926 cells under fluorescence microscope (upper) or microscope (lower) (magnification × 100). Representative pictures were captured in three independent experiments. b Cell count assay to evaluate the adherent THP-1 cells. *P < 0.01. Scale bar = 100 μm.

Fig. 4

Colocalization of CD74 and CXCR4 in EA.hy926 cells infected with P. gingivalis. EA.hy926 cells infected with P. gingivalis (24 h, MOI = 100) were observed by fluorescence microscopy (bottom). The cells cultured with medium alone were used as a control (top). Colocalization of CD74 and CXCR4 in the plasma membrane (orange-yellow overlay) was shown. After three independent experiments, representative pictures were captured by fluorescence microscope (magnification × 400). Scale bar = 20 μm.

Fig. 5

Receptor complex formation between CD74 and CXCR4 for MIF in EA.hy926 cells infected with P. gingivalis. Input controls (lysates without immunoprecipitation) are shown (a). Coimmunoprecipitation and pull-down assays were performed by anti-MIF antibody (b) or anti-IgG antibody (c) for immunoprecipitation (IP), and then anti-MIF antibody, anti-CXCR4 antibody, and anti-CD74 antibody were used for Western blot (WB). A: EA.hy926 cells infected withP. gingivalis for 24 h (MOI = 100). B: EA.hy926 cells stably transfected with empty vectors and infected with P. gingivalis. C: EA.hy926 cells stably transfected with CXCR4-silencing plasmid and infected with P. gingivalis. Data were representative of three independent experiments.