GPR4 cells (Fig. 5C). In contrast, overexpression of the GPR4 R115A mutant failed to further augment the mRNA levels of these adhesion molecules upon acidosis stimulation in HUVEC cells (Figs. 5A, B and C). These results are in accordance with the U937 monocyte adhesion assay results (Figs. 1B and 2B). Immunofluorescence staining was performed to assess the expression of E-selectin, VCAM-1, and ICAM-1 at the protein level. When HUVEC/Vector and HUVEC/GPR4 cells were treated with pH 8.4 and pH 7.4, the expression of E-selectin, VCAM-1, and ICAM-1 was at the basal level. Acidic pH 6.4 treatment stimulated the expression of E-selectin and ICAM-1 in HUVEC/Vector cells and the expression was further increased in HUVEC/GPR4 cells (Fig. 6). While VCAM-1 was barely detectable by this antibody in HUVEC/Vector cells, its expression was clearly up-regulated by pH 6.4 in HUVEC/GPR4 cells (Fig. 6). We also examined whether acidosis/GPR4 could induce the expression or membrane translocation of P-selectin, another adhesion molecule in endothelial cells [55,56,57]. The real-time RT-PCR result did not show any significant up-regulation of P- selectin mRNA in HUVECs induced by acidosis/GPR4 (Fig. S4A). Moreover, we could not detect cell membrane translocation of P-selectin protein from the Weibel-Palade body by the acidosis treatment for 5 h (Fig. S4B) or 20 min (data not shown). In comparison, the thrombin treatment for 20 min as a positive control induced the translocation of P-selectin to cell membrane (Fig. S4B). Collectively, the results suggest that activation of GPR4 by acidosis in HUVEC cells increases the expression of multiple endothelial adhesion molecules including E-selectin, VCAM-1 and ICAM-1, which may interact with their receptors on U937 cells to mediate the adhesion. Blockade of endothelial E-selectin, VCAM-1, and ICAM-1 on HUVECs reduces acidosis/GPR4-mediated cell adhesion Monoclonal blocking antibodies were used to examine whether E-selectin, VCAM-1, and ICAM-1 are functionally involved in acidosis/GPR4-induced HUVEC adhesion. After the treatment with different pHs for 5 h, HUVEC/GPR4 cells were incubated with E-selectin, VCAM-1, ICAM-1 blocking antibodies or the mouse IgG1 control and the static cell adhesion assay was performed. Compared to the IgG1 control, each of the three blocking antibodies significantly decreased the adhesion of HUVECs with U937 monocytes (Fig. 7). It was also noted that the ICAM-1 antibody caused an increase of basal adhesion for unknown reasons. The blocking antibody results suggest that multiple adhesion molecules, including E-selectin, VCAM-1 and ICAM-1, are functionally important for acidosis/GPR4-induced HUVEC adhesion with U937 monocytes. Hypercapnic acidosis can also activate GPR4 to increase the adhesion molecule expression and the adhesiveness of HUVECs In all the results described above, HUVECs were treated with isocapnic acidosis in which EGM-2 media were buffered with HEPES, EPPS and MES and the acidic pH was obtained by adding HCl. Hypercapnic acidosis, caused by an increase in carbon dioxide in tissues, blood or body fluid, is another type of acidosis that is associated with many pathological conditions [9,11]. To investigate the effects of hypercapnic acidosis on GPR4 signaling, HUVEC/Vector and HUVEC/GPR4 cells were treated with EGM-2 culture media under ambient air (,0.04% CO2), 5% CO2 and 20% CO2. The pHs of the EGM-2 media Acidosis/GPR4 and Endothelial Cell Adhesion Figure 7. Blockade of E-selectin, VCAM-1 or ICAM-1 reduces the adhesion of HUVECs with U937 monocytic cells. HUVECs stably overexpressing GPR4 were treated with different pHs as indicated for 5 to 15 h, followed by 30 min incubation with pH 7.4 medium containing 5 mg/ml IgG1 control antibody, anti-E-selectin, anti-VCAM-1, or anti-ICAM-1 antibody. The cell adhesion assay was then performed as described under ‘‘Materials and Methods’’. **, P,0.01; ***, P,0.001; compared with the IgG1 control group at pH 6.4. The results are representative of two independent experiments. Error bars are the mean 6 SEM. doi:10.1371/journal.pone.0027586.g007 under these levels of CO2 were around 8.4, 7.4 and 6.4, respectively. As measured by real-time RT-PCR, the hypercapnic acidosis treatment (20% CO2), in comparison to 5% CO2 and ambient air, increased the expression of E-selectin, VCAM-1 and ICAM-1 mRNA by several fold in HUVEC/Vector cells, and the expression was further substantially induced in HUVEC/GPR4 cells but not HUVEC/GPR4 R115A mutant cells (Figs. 8A, B and C). Moreover, the hypercapnic acidosis (20% CO2) treatment stimulated the production of cAMP in HUVEC/Vector cells, with further increase in HUVEC/GPR4 cells (Fig. 8D). Concordantly, the hypercapnic treatment augmented the adhesiveness of HUVEC/Vector cells, and this effect was particularly robust in HUVEC/GPR4 cells (Fig. 8E). These results suggest that, similar as isocapnic acidosis, hypercapnic acidosis can also activate GPR4 to induce the expression of the endothelial adhesion molecules, stimulate cAMP production, and increase HUVEC adhesiveness. Discussion Acidosis has been shown to regulate vascular tone, blood vessel growth, and the function of smooth muscle and endothelial cells [12,58,59]. At the molecular level, ATP-sensitive potassium channels, activated by intracellular acidic pH in vascular smooth muscle cells, are involved in acidosis-induced coronary arteriolar dilation [60,61]. Recent studies indicate that extracellular acidosis activates acid-sensing ion channels (ASIC) to regulate the function of smooth muscle cells [62]. We have recently shown that extracellular acidosis activates GPR4 in endothelial cells and leads to cAMP production and the inhibition of endothelial cell migration and microvessel growth [15], suggesting that GPR4 is a functional pH sensor in endothelial cells. In this report, we have identified a novel function of the GPR4 receptor. Activation of GPR4 by acidosis enhances HUVEC adhesiveness and binding with U937 monocytes. This effect is augmented by GPR4 overexpression and diminished by GPR4 knockdown in HUVECs. Since the interaction between endothe- lial cells and leukocytes is critical for inflammatory response, acidosis/GPR4 signaling may represent a new pathway in inflammation. With particular relevance, local interstitial acidosis is commonly found in inflammatory tissues. For instance, acidic tissue pH is associated with asthma, arthritis, and cystic fibrosis [2,4,6]. Acidosis is also frequently identified in ischemia and solid PLoS ONE | www.plosone.org 10 November 2011 | Volume 6 | Issue 11 | e27586