Activation of p38 MAPKα by extracellular pressure mediates the stimulation of macrophage phagocytosis by pressure

Abstract

We have previously demonstrated that constant 20 mmHg extracellular pressure increases serumopsonized latex bead phagocytosis by phorbol 12-myristate 13-acetate (PMA)-differentiated THP-1 macrophages in part by inhibiting focal adhesion kinase (FAK) and extracellular signal-regulated kinase (ERK). Because p38 MAPK is activated by physical forces in other cells, we hypothesized that modulation of p38 MAPK might also contribute to the stimulation of macrophage phagocytosis by pressure. We studied phagocytosis in PMA-differentiated THP-1 macrophages, primary human monocytes, and human monocyte-derived macrophages (MDM). p38 MAPK activation was inhibited using SB-203580 or by p38 MAPK␣ small interfering RNA (siRNA). Pressure increased phagocytosis in primary monocytes and MDM as in THP-1 cells. Increased extracellular pressure for 30 min increased phosphorylated p38 MAPK by 46.4 Ϯ 20.5% in DMSO-treated THP-1 macrophages and by 20.9 Ϯ 9% in primary monocytes (P Ͻ 0.05 each). SB-203580 (20 M) reduced basal p38 MAPK phosphorylation by 34.7 Ϯ 2.1% in THP-1 macrophages and prevented pressure activation of p38. p38 MAPK␣ siRNA reduced total p38 MAPK protein by 50 -60%. Neither SB-203580 in THP-1 cells and peripheral monocytes nor p38 MAPK siRNA in THP-1 cells affected basal phagocytosis, but each abolished pressure-stimulated phagocytosis. SB-203580 did not affect basal or pressure-reduced FAK activation in THP-1 macrophages, but significantly attenuated the reduction in ERK phosphorylation associated with pressure. p38 MAPK␣ siRNA reduced total FAK protein by 40 -50%, and total ERK by 10 -15%, but increased phosphorylated ERK 1.4 Ϯ 0.1-fold. p38 MAPK␣ siRNA transfection did not affect the inhibition of FAK-Y397 phosphorylation by pressure but prevented inhibition of ERK phosphorylation. Changes in extracellular pressure during infection or inflammation regulate macrophage phagocytosis by a FAKdependent inverse effect on p38 MAPK␣ that might subsequently downregulate ERK.force; inflammation; infection; leukocyte; mechanotransduction; signal transduction MONOCYTES AND MACROPHAGES are recruited to sites of inflammation and play critical roles in innate host defense mechanisms. Tissue pressure is often altered in association with inflammation or infection. Mechanical stimuli such as pressure are known to modulate cellular morphology and function in other cell types (7,38,39,73,79). We have previously reported that constant low extracellular pressure increases phagocytosis by phorbol 12-myristate 13-acetate (PMA)-differentiated THP-1 macrophages (67). Although focal adhesion kinase (FAK) and extracellular signal-regulated kinase (ERK) are activated by physical force caused by pressure in some other cell types (2,4,44,75,76,80), the effect of pressure on macrophage phagocytosis appears partially mediated by inhibition of a FAK-ERK signal pathway (67). Like ERK, the mitogen-activated protein kinase (MAPK) p38 is activated by various stress stimuli, including lipopolysaccharide (LPS) stimulation in macrophages and physical force...

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

“…Exposure to pressure increased phagocytosis in monocytes from healthy volunteers (22.9 Ϯ 3.3 vs. 31.8 Ϯ 4.7%, n ϭ 8, P Ͻ 0.005; Fig. 1A), consistent with our previous observations (51,52). However, pressure failed to increase phagocytosis in monocytes isolated from surgical patients receiving anesthesia (Fig.…”

“…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).…”

“…Peripheral blood mononuclear cells were isolated from heparinized venous blood by Ficoll-Hypaque densitygradient sedimentation as described previously (51). Peripheral blood mononuclear cells were suspended at a density of 5 ϫ 10 6 /ml in RPMI 1640 medium supplemented with penicillin G (100 U/ml), 2 mM L-glutamine, and 5% fresh unheated human serum.…”

“…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.…”

“…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.…”