Hp induces CCR2 internalization in monocytes As with other chemokines with agonistic activity, CCR2 activation is followed by internalization and several research reported CCR2 disappearance from cell surface following exposure to MCP1. To further prove the possible interaction between CCR2 and Hp, we studied the impact of Hp on CCR2 internaliza tion. MCP1 and Hp pretreatments induced a dose dependent disappearance of CCR2 receptor from the surface of U937 cells as assessed by flow cytometric analysis. A comparable, albeit less dramatic impact was observed in pri mary monocytes. Differences in CCR2 surface expression could partly account for the significantly less pronounced internalization observed in these cells. Indeed, when assessed by flow cytometry major monocytes displayed on average around 50% in the CCR2 surface expression found in U937 cells.
We next wanted to rule out the possibility that the observed CCR2 disappearance from cell surface was resulting from Hp interference with CCR2 binding to its antibody. To this end U937 cells had been treated with 0. 5 mg ml Hp or BSA, fixed, permeabilized and stained. When when compared with cells similarly treated with Hp but not permeabilized, these selleckchem samples showed a 50% improve in CCR2 expression. This can be a further indication that Hp induces CCR2 internalization. Hp promotes CCR2 signaling The MAPK signal transduction pathway is activated in response to the interaction of CCR2 with ligand, and irrespective of whether this pathway is implicated in the cellular events leading to chemotaxis is a topic of debate.
To search for extra evidence that Hp is in a position to activate CCR2 we assessed the phosphorylation state of extracellular sig nal regulated kinase 1 2 in U937 cells previously starved overnight selleck MEK Inhibitor and subsequently incu bated with Hp, with MCP1, or basically stimulated with 10% serum. As shown within the immunoblot and bar graph of Figure 7 there was a important induction of ERK1 two phosphorylation inside the Hp treated sample, the intensity of the signal being comparable to that observed for the MCP1 treated samples. When cells had been treated together with the CCR2 antagonist RS102895 a dramatic lower in ERK1 2 phosphorylation was observed in the cells treated with Hp and with MCP1, but not in those serum stimu lated. Conversely, ERK1 two phosphorylation was abol ished in all types of therapy when U0126, the selective inhibitor with the ERK upstream kinase MAP ERK kinase was employed.
To further explore the capability of Hp to activate the ERK1 two pathway, a chemotaxis assay employing Hp and MCP1 as chemotactic agents was performed with U937 cells previously incubated with U0126. The results of this experiment, summarized within the bar graph of Figure 8a, indicate that blocking the ERK1 two pathway final results within a dramatic reduction in the capability of This result needs to be thought of in the light of clinical studies on diabetic individuals that indicate an association among the presence on the Hp 2 two phenotype along with a more frequent onset of complications and cardiovascular illness.