Extracellular pressure stimulates macrophage phagocytosis by inhibiting a pathway involving FAK and ERK

Abstract

We hypothesized that changes in extracellular pressure during inflammation or infection regulate macrophage phagocytosis through modulating the focal adhesion kinase (FAK)-ERK pathway. Undifferentiated (monocyte-like) or PMA-differentiated (macrophage-like) THP-1 cells were incubated at 37 degrees C with serum-opsonized latex beads under ambient or 20-mmHg increased pressure. Pressure did not affect monocyte phagocytosis but significantly increased macrophage phagocytosis (29.9 +/- 1.8 vs. 42.0 +/- 1.6%, n = 9, P < 0.001). THP-1 macrophages constitutively expressed activated FAK, ERK, and Src. Exposure of macrophages to pressure decreased ERK and FAK-Y397 phosphorylation (77.6 +/- 7.9%, n = 7, P < 0.05) but did not alter FAK-Y576 or Src phosphorylation. FAK small interfering RNA (SiRNA) reduced FAK expression by >75% and the basal amount of phosphorylated FAK by 25% and significantly increased basal macrophage phagocytosis (P < 0.05). Pressure inhibited FAK-Y397 phosphorylation in mock-transfected or scrambled SiRNA-transfected macrophages, but phosphorylated FAK was not significantly reduced further by pressure in cells transfected with FAK SiRNA. Pressure increased phagocytosis in all three groups. However, FAK-SiRNA-transfected cells exhibited only 40% of the pressure effect on phagocytosis observed in scrambled SiRNA-transfected cells so that phagocytosis inversely paralleled FAK activation. PD-98059 (50 microM), an ERK activation inhibitor, increased basal phagocytosis (26.9 +/- 1.8 vs. 31.7 +/- 1.1%, n = 15, P < 0.05), but pressure did not further increase phagocytosis in PD-98059-treated cells. Pressure also inhibited ERK activation after mock transfection or transfection with scrambled SiRNA, but transfection of FAK SiRNA abolished ERK inhibition by pressure. Pressure did not increase phagocytosis in MonoMac-1 cells that do not express FAK. Increased extracellular pressure during infection or inflammation enhances macrophage phagocytosis by inhibiting FAK and, consequently, decreasing ERK activation.

Propofol inhibits pressure-stimulated macrophage phagocytosis via the GABAA receptor and dysregulation of p130cas phosphorylation

“…Propofol does not act via the signal pathways previously reported to mediate the pressure effect on phagocytosis. Previous studies demonstrated that decreasing FAK and ERK activation and increasing the activation of p38 and the PI3K-Akt-p70S6k pathway contribute to the stimulation of macrophage phagocytosis by extracellular pressure (51)(52)(53). We therefore examined the effect of propofol on these signals.…”

“…We previously reported that increased extracellular pressure (20 mmHg above ambient pressure) stimulates serum-opsonized latex bead uptake by primary isolated monocytes from healthy volunteers as well as PMA-differentiated human monocytic THP-1 cells (THP-1 macrophages) (51)(52)(53). Several reports suggest that anesthetic agents and surgical stress may affect phagocytic cell function (5,23,28) and that tissue pressure may be increased within surgical wounds or at surgical sites postoperatively (56).…”

“…PMA-differentiated THP-1 cells (1 ϫ 10 6 ) in 1 ml of tissue culture medium supplemented with 10% FBS in a 35-mm petri dish were mixed with opsonized latex beads at a multiplicity ratio of 1:5 and then incubated for 2 h at 37°C. One set of dishes was placed in a calibrated pressure box described previously (51,53). The pressure inside the box was set at 20 mmHg, and the pressure box was placed inside an incubator at 37°C and monitored every 15 min.…”

“…At 24 -48 h after transfection, cells were used for Western analysis and assays of phagocytosis. Transfection efficacy, measured by transfection of Cy3-conjugated siRNA targeted to luciferase (Upstate, Charlottesville, VA), was 90 -97% (53).…”

“…We previously reported that exposure to constant low extracellular pressure (20 mmHg) enhances phagocytosis by human peripheral monocytes and PMA-differentiated THP-1 macrophages through activation of p38 and the phosphatidylinositol 3-kinase (PI3K)-Akt-p70S6k pathway and by inhibition of focal adhesion kinase (FAK) and ERK (51)(52)(53). This is important because interstitial tissue pressure may increase at wound sites after surgery.…”