Activation of p38 MAPK{alpha} by extracellular pressure mediates the stimulation of macrophage phagocytosis by pressure

Hiroe Shiratsuchi and Marc D. Basson

Department of Surgery, School of Medicine, Wayne State University and John D. Dingell Veterans Affairs Medical Center, Detroit, Michigan

Submitted 9 November 2004 ; accepted in final form 27 December 2004


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
We have previously demonstrated that constant 20 mmHg extracellular pressure increases serum-opsonized 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{alpha} 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 ± 20.5% in DMSO-treated THP-1 macrophages and by 20.9 ± 9% in primary monocytes (P < 0.05 each). SB-203580 (20 µM) reduced basal p38 MAPK phosphorylation by 34.7 ± 2.1% in THP-1 macrophages and prevented pressure activation of p38. p38 MAPK{alpha} 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{alpha} siRNA reduced total FAK protein by 40–50%, and total ERK by 10–15%, but increased phosphorylated ERK 1.4 ± 0.1-fold. p38 MAPK{alpha} 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 FAK-dependent inverse effect on p38 MAPK{alpha} 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 forces in various other cell types (43, 44, 53, 55, 58, 72, 75, 76). We therefore hypothesized that changes in extracellular pressure might modulate p38 MAPK activation and that such modulation might contribute to the regulation of macrophage phagocytosis by changes in extracellular pressure during inflammation or infection.

Recent reports suggest that physical forces such as extracellular pressure and repetitive strain may alter other aspects of macrophage functions. Pressure (40–130 mmHg) increases monocyte migration in a dose-dependent manner and enhances scavenger receptor expression in macrophages. Macrophages produce proinflammatory cytokines in response to very high cyclic pressure (>1,000 mmHg), the combination of cyclic strain and 75 mmHg pressure, or high pressure (>70 mmHg) combined with stimulation by endotoxin. Increased pressure (40–90 mmHg), but not cyclic strain, has also been shown to increase the uptake of aggregated IgG by the mouse macrophage J774.16 cell line (46, 47, 52). The mechanisms or intracellular signals responsible for this effect have not been defined.

However, these pressures are higher than are often observed in edematous tissue. We therefore examined the effects of constant low pressure (20 mmHg) on macrophage phagocytosis by studying human primary isolated peripheral monocytes and monocyte-derived macrophages (MDM) as well as PMA-differentiated (macrophage-like) human THP-1 monocytic cells in which we had previously described pressure-increased phagocytosis (67).

p38 MAPK is activated by microbial phagocytosis in phagocytic cells (50, 58) and in response to mechanical stimuli, such as pressure and strain in some other types of cells (2, 4, 44, 75, 76, 80), and activates downstream kinases mediating inflammatory reactions (3, 55, 72). In this study, we examined the involvement of p38 MAPK activation by pressure in THP-1 macrophage and human monocyte phagocytosis. In further studies, we explored the relationship between the previously described inhibition of FAK and ERK by pressure and the p38 MAPK activation by pressure that we now report. Although a relationship between FAK and ERK and integrin-mediated phagocytosis in macrophages has not previously been established, FAK and ERK are activated by integrin cross-linking after phagocytosis occurs (48, 61), and downregulation of the FAK-ERK signal partially mediates pressure-induced macrophage phagocytosis (67). We used the pharmacological inhibitor SB-203580 to probe the role of p38 MAPK in mediating the effect of pressure, as well as a specific small interfering RNA (siRNA) targeted to the p38 MAPK{alpha} isoform.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Antibodies and chemicals. Antibodies against total p38 MAPK, phospho-p38 MAPK, total p38 MAPK{alpha}, total p38 MAPK{delta}, total ERK, phospho-ERK, and phospho-Src were purchased from Cell Signaling Technology (Beverly, MA). Anti-total FAK and anti-pY397-FAK antibodies were obtained from Upstate USA (Charlottesville, VA) and BioSource International (Camarillo, CA), respectively. Fluorescence-labeled latex beads (2.0 µm) were obtained from Polysciences (Warrington, PA). SB-203580, a specific inhibitor for p38 MAPK{alpha} and p38 MAPK{beta}, was obtained from Calbiochem (Santa Cruz, CA).

Cells and cell cultures. The human monocytic cell line THP-1 was obtained from the American Type Culture Collection (Rockville, MD) and maintained in RPMI 1640 (GIBCO, Grand Island, NY) supplemented with 10% FBS (Sigma, St. Louis, MO), L-glutamine (200 mM), and 2-mercaptoethanol (5 x 10–5 M; Sigma). THP-1 cells (5 x 105 cells/35-mm dish) were differentiated by stimulation with PMA (50 ng/ml, final concentration) for 3 days to obtain a macrophage-like phenotype that closely resembles human MDM, as previously reported (5, 14, 74).

Peripheral venous blood was obtained from consenting healthy volunteers by a protocol approved by the Human Investigation Committee of Wayne State University School of Medicine and the John D. Dingell Veterans Affairs Medical Center. Peripheral blood mononuclear cells (PBMC) were isolated from heparinized venous blood by Ficoll-Hypaque density gradient sedimentation, as described previously (66, 68, 69). PBMC were suspended at a density of 1 x 107/ml in RPMI 1640 medium supplemented with 100 U/ml penicillin G, 2 mM L-glutamine, and 10% fresh autologous unheated human serum. One 100-µl droplet of cell suspension was plated in each well of a LabTek chamber. The chambers then were incubated for 60 min at 37°C. Nonadherent cells were washed off, and the monocyte monolayers were used for phagocytosis assays. MDM were prepared by culturing monocytes for 6 days in RPMI 1640 medium with 10% autologous unheated serum at 37°C in 5% CO2.

Pressure box model. Ambient pressure was controlled by an apparatus previously used in other studies (7, 67, 73, 80). Cells are placed in an air-tight Lucite chamber with gas inlet and outlet valves, thumb screws, and an O-ring to achieve an airtight seal and a pressure manometer to monitor inside pressure. The box was prewarmed to 37°C for 1 h before each study to prevent fluctuations in internal pressure caused by temperature shifts of the pressurizing gas; the appropriate target pressure was achieved within 1 min. Temperature and pressure were maintained within 2°C for temperature and 1.5 mmHg for pressure using this method, and the pH, osmolarity, and PO2 of the culture medium remained essentially the same over the course of the study (7).

Assay for phagocytosis. Fluorescence-labeled latex beads (2.0 µm) were opsonized with 10% unheated FBS for 60 min at 37°C before the experiments. PMA (1 x 106)-differentiated THP-1 in 1 ml of tissue culture medium supplemented with 10% FBS in a 35-mm petri dish, 1 x 105 primary isolated human monocyte monolayers, or MDM in 500 µl of RPMI 1640 with 10% autologous unheated serum in a LabTek chamber were mixed with opsonized latex beads at a multiplicity ratio of 1:5 and incubated for 2 h at 37°C. One set of dishes was placed in a calibrated pressure box. The pressure inside the pressure box was set at 20 mmHg, and the pressure box was placed inside an incubator at 37°C and monitored every 15 min. The second set of dishes was placed inside the same incubator but under ambient pressure conditions. After 2 h, macrophage monolayers were washed vigorously with PBS to remove extracellular beads, fixed with methanol for 10 min, and then counterstained with methylene blue. The number of intracellular latex particles was determined by counting fluorescent beads within cells under a fluorescence microscope. Data were expressed as percent phagocytosis, calculated as the total number of cells with at least one bead as a percentage of the total number of cells counted.

Western immunoblotting. THP-1 macrophages were incubated under ambient or increased pressure (20 mmHg) conditions for 30 min, rinsed one time with cold PBS, and lysed with lysing buffer. Protein concentrations in cell lysates were measured using the bicinchoninic acid protein assay reagent (Pierce, Rockford, IL). Equal amounts of protein were loaded in each lane. Cell lysates were resolved under reducing conditions by 10% SDS-PAGE and then transferred to nitrocellulose membranes. After being blocked with 5% BSA in Tris-buffered saline (TBS) with 0.1% Tween 20, the membranes were incubated with antibodies against activated or total forms of protein overnight at 4°C, washed three times with 0.1% Tween 20-TBS, and then incubated for 60 min with 2,000:1 peroxidase-conjugated anti-rabbit IgG. The membrane-bound peroxidase activity was detected using ECL Plus Western Blotting Detection kits (Amersham, Arlington Heights, IL). Chemiluminescent images were captured and analyzed using a Kodak Digital Science Image Station 440CF. All blots were studied within the linear range of exposure.

Transfection of siRNA. To inhibit p38 MAPK or FAK protein expression, THP-1 cells were transfected with siRNA. The siRNA targeted to p38 MAPK{alpha} was purchased from Upstate USA (catalog no. M-003556). The siRNA duplex targeted to FAK, 5'-GCAUGUGGCCUGCUAUGGAdTdT/dTdTCGUACACCGGACGAUACCU-5', directed toward the mRNA target, 5'-AAGCAUGUGGCCUGCUAUGGA-3', has been previously described (67, 73) and was synthesized by Dharmacon (Lafayette, CO). siRNA targeted to p38 MAPK and a scrambled RNA duplex used as a control were purchased from Dharmacon. THP-1 cells (4 x 105 cells/60-mm petri dish) were stimulated with PMA (50 ng/ml) for 2 days before transfection. Transfection of duplex siRNAs were performed using Oligofectamine (GIBCO, Gaithersburg, MD) according to the manufacturer's protocol. After transfection (24 h), cells were used for Western analysis and for assays of phagocytosis. Transfection efficacy, measured by transfecting Cy3-conjugated siRNA targeted to luciferase (Upstate USA), was 90–97% (67).

Statistical analysis. The significance of differences between groups was calculated by the Student's t-test or paired t-test as appropriate. Confidence (95%) was set a priori as the desired level of statistical significance.


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Pressure effect on primary human cells. Previously we reported that extracellular constant low pressure (20 mmHg) increases PMA-differentiated THP-1 macrophage phagocytosis but not undifferentiated THP-1 monocytes (67). We now sought to determine whether primary isolated human phagocytes responded to extracellular pressure similarly to the cell line we had studied previously (67). Indeed, exposure to pressure for 2 h significantly increased latex bead uptake by monocytes (23.7 ± 4.7 vs. 31.9 ± 6.3%, P < 0.01, paired t-test; Fig. 1A) and MDM (32.3 ± 7.8 vs. 41.9 ± 8.2, P < 0.005, paired t-test) as well as THP-1 macrophages (Fig. 1B).



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Fig. 1. Pressure effect on phagocytosis. A: pressure effect on phagocytosis by primary human mononuclear cells. Primary isolated human peripheral monocytes (MN) or monocyte-derived macrophages (MDM) were incubated with serum-opsonized fluorescence-labeled latex beads (5 beads/cell) under ambient pressure or 20 mmHg increased pressure conditions for 2 h. Cells were then washed with PBS and fixed. The number of intracellular latex beads was counted using a fluorescence microscope. Data were expressed as %phagocytosis calculated as detailed in MATERIALS AND METHODS. Pressure increased phagocytosis by primary human monocytes (P < 0.01, n = 6) and MDM (P < 0.005, n = 4). B: pressure effect on THP-1 macrophage phagocytosis. Phorbol 12-myristate 13-acetate (PMA)-differentiated THP-1 macrophages were incubated with serum-opsonized fluorescence-labeled latex beads (5 beads/cell) under ambient pressure or 20 mmHg increased pressure conditions for 2 h. Cells were then washed with PBS and fixed. The number of intracellular latex beads was counted using a fluorescence microscope. Data were expressed as %phagocytosis calculated as detailed in MATERIALS AND METHODS. Pressure increased THP-1 macrophage phagocytosis (*P < 0.001, n = 17).

 
Potential role of p38 MAPK. We next examined the potential role of p38 MAPK in pressure-mediated phagocytosis. Exposure to pressure for 30 min increased p38 MAPK activation by 46.4 ± 20.5% (P < 0.05, n = 7) in THP-1 macrophages and by 20.8 ± 2.7% (P < 0.02, n = 4 paired t-test) in primary human monocytes (Fig. 2A). Pretreatment of THP-1 macrophages with 20 µM SB-203580 for 60 min before exposure to pressure inhibited basal p38 MAPK phosphorylation by 34.7 ± 2.1% (P < 0.02) and blocked the activation of p38 MAPK by pressure compared with cells treated with a DMSO vehicle control (Fig. 2B). Phagocytosis in THP-1 macrophages after exposure to increased pressure for 2 h was significantly increased compared with cells exposed to ambient pressure in cells treated with a DMSO vehicle control (P < 0.001, n = 15), and basal rates of phagocytosis were not significantly altered by pretreatment with SB-203580. However, SB-203580 pretreatment essentially ablated the stimulation of phagocytosis by pressure compared with DMSO controls (P < 0.005, n = 15; Fig. 2C). Pretreatment with SB-203580 similarly abrogated the stimulation of phagocytosis by pressure in human peripheral monocytes (Fig. 2D).



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Fig. 2. Studies of p38 MAPK and pressure. A: increase in p38 MAPK phosphorylation in primary human monocytes and THP-1 macrophages by pressure. Top: typical Western blots for phosphorylated p38 MAPK, each stripped and reprobed for total p38 MAPK as a loading control. Histograph summarizes densitometric results expressed as means ± SE of the ratio of phosphorylated p38 MAPK to total p38 MAPK, normalized to control. Open bars, ambient pressure; filled bars, increased pressure. Increased pressure stimulated p38 MAPK phosphorylation in primary human monocytes (*P < 0.05, n = 4) and in THP-1 macrophages (*P < 0.05, n = 7). B: inhibition of p38 MAPK phosphorylation in PMA-differentiated THP-1 macrophages by SB-203580. Top: typical Western blot for phosphorylated p38 MAPK, stripped and reprobed for total p38 MAPK as a loading control, in cells pretreated with DMSO vehicle or SB-203580 under control or increased pressure conditions. Histograph summarizes densitometric results expressed as means ± SE of the ratio of phosphorylated p38 MAPK to total p38 MAPK, normalized to DMSO vehicle control. Open bars, ambient pressure; filled bars, increased pressure. Pressure increased p38 MAPK phosphorylation (*P < 0.05, n = 5). Pretreatment of PMA-differentiated THP-1 cells with SB-203580 (20 µM) blocked p38 MAPK phosphorylation (#P < 0.05, n = 5) and abolished the effect of pressure, but treatment with DMSO vehicle control had no effect. C: effect of SB-203580 on PMA-differentiated THP-1 macrophage phagocytosis under ambient and increased pressure conditions. Results are expressed as means ± SE of %phagocytosis in PMA-differentiated THP-1 cells pretreated with SB-203580 or DMSO vehicle. Open bars, ambient pressure; filled bars, increased pressure. Pretreatment with SB-203580 did not affect basal phagocytosis but abrogated pressure-induced phagocytosis. D: effect of SB-203580 on primary human monocyte phagocytosis under ambient and increased pressure conditions. Results are expressed as means ± SE of %phagocytosis in PMA-differentiated THP-1 cells pretreated with SB-203580 or DMSO vehicle. Open bars, ambient pressure; filled bars, increased pressure. Pretreatment with SB-203580 did not affect basal phagocytosis but prevented pressure-induced phagocytosis. NS, not significant.

 
To further examine whether the augmentation of p38 MAPK activation by pressure might contribute to pressure-induced phagocytosis, siRNA targeted to p38 MAPK{alpha} was transfected in PMA-stimulated THP-1 cells. As shown in Fig. 3, A and B, transfection with p38 MAPK{alpha} siRNA in THP-1 macrophages reduced total p38 MAPK{alpha} by 50–70%, but p38 MAPK{delta} by only 10%, compared with cells transfected with scrambled siRNA. Cells transfected with p38 MAPK{alpha} siRNA tended to exhibit a slightly decreased basal phagocytic activity compared with THP-1 macrophages transfected with scrambled siRNA. However, this result did not achieve statistical significance. Transfection of p38 MAPK{alpha} siRNA completely abolished pressure-induced phagocytosis (Fig. 3C). Exposure to pressure increased p38 MAPK phosphorylation in THP-1 macrophages transfected with scrambled siRNA. Transfection with the siRNA targeted to p38 MAPK{alpha} tended to increase the relative proportion of activation in the residual p38 MAPK protein. However, the total amount of activated p38 MAPK was markedly reduced in these p38 MAPK{alpha} siRNA-transfected cells, and no effect of pressure on p38 MAPK activation could be demonstrated (data not shown).



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Fig. 3. Transfection with small interfering RNA (siRNA) targeted to p38 MAPK{alpha} in PMA-differentiated THP-1 macrophages. A: specificity of siRNA. Typical Western blots probed for antibodies against total p38 MAPK{alpha}/{beta}, total p38 MAPK{alpha}, total p38 MAPK{delta}, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a loading control. Transfection with p38 MAPK{alpha} decreased p38 MAPK{alpha} but not p38 MAPK{delta} expression. SC, cells transfected with scrambled siRNA; p38{alpha}, cells transfected with siRNA targeted to p38 MAPK{alpha}. Data are representative of at least 3 typical blots. B: total p38 MAPK protein expression in cells transfected with scrambled siRNA or p38 MAPK{alpha} siRNA incubated under ambient or increased pressure conditions. Top: typical Western blot for total p38 MAPK, stripped and reprobed for GAPDH as a loading control. Histograph summarizes densitometric analyses, expressed as mean ± SE of total p38 MAPK to GAPDH, normalized to control scrambled siRNA-transfected cells. Open bars, ambient pressure; filled bars, increased pressure. Total p38 MAPK protein in cells transfected with p38 MAPK{alpha} siRNA was significantly lower than p38 MAPK protein in scrambled siRNA-transfected cells (*P < 0.01, n = 3). C: effect of pressure on phagocytosis in cells transfected with scrambled siRNA and cells transfected with p38 MAPK{alpha} siRNA. Results are expressed as mean ± SE of %phagocytosis. Open bars, ambient pressure; filled bars, increased pressure. Transfection with p38 MAPK{alpha} siRNA did not affect THP-1 macrophage phagocytosis under ambient pressure. However, pressure did not stimulate phagocytosis in cells transfected with p38 MAPK{alpha} siRNA. *P < 0.05 compared with ambient pressure control and p38 MAPK{alpha} siRNA transfected under increased pressure.

 
Effect of FAK silencing by siRNA on p38 MAPK expression and activation by pressure. Our previous study demonstrated that reduction of FAK protein by transfection of FAK siRNA increases THP-1 macrophage phagocytosis (67). It therefore became worthwhile to determine whether modulating FAK influenced p38 activation. Accordingly, we next examined p38 MAPK activation in THP-1 macrophages transfected with FAK siRNA. Transfection of FAK siRNA did not alter total p38 protein expression (Fig. 4, blot on bottom). Pressure significantly increased p38 MAPK phosphorylation in THP-1 macrophages transfected with scrambled siRNA. Basal p38 activation tended to increase in response to transfection with siRNA targeted to FAK, although this did not achieve statistical significance. However, pressure-induced p38 MAPK phosphorylation was abrogated in THP-1 macrophages transfected with FAK siRNA (Fig. 4). These data suggest that FAK signaling might stimulate basal p38 activation but prevent a further increase in p38 MAPK activation by pressure.



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Fig. 4. Effect of pressure on p38 MAPK activation in PMA-differentiated THP-1 macrophages transfected with focal adhesion kinase (FAK) siRNA. Top: typical Western blot for phosphorylated p38 MAPK, stripped and reprobed for total p38 MAPK as a loading control. Histograph summarizes densitometric analyses, expressed as means ± SE of phosphorylated p38 MAPK to total p38 MAPK normalized to control. Open bars, ambient pressure; filled bars, increased pressure. Pressure increased p38 MAPK phosphorylation in scrambled siRNA-transfected cells (*P < 0.05, n = 5) but did not alter p38 MAPK phosphorylation in cells transfected with FAK siRNA to inhibit FAK.

 
Effect of ERK inhibition by PD-98059 on p38 MAPK activation. We examined whether inhibition of ERK phosphorylation by pharmacological inhibitor PD-98059 might modulate enhancement of p38 MAPK activation by pressure. Pretreatment of THP-1 macrophages with PD-98059 inhibited ERK phosphorylation but did not affect p38 MAPK phosphorylation at ambient pressure conditions or pressure-induced increased p38 MAPK phosphorylation (data not shown).

Effect of p38 MAPK inhibition on FAK and ERK. We next examined the converse, whether the effect of pressure on p38 MAPK activation might modulate the inhibition of FAK and ERK signals previously shown to contribute to the pressure effect. In initial studies, p38 MAPK was inhibited by pretreatment with 20 µM SB-203580 for 60 min before exposure to pressure. FAK and ERK activation by pressure were then examined. Pretreatment of THP-1 macrophages with SB-203580 did not alter total FAK or ERK protein expression (see blots on bottom in Fig. 5, A and B). SB-203580 pretreatment significantly increased ERK phosphorylation and prevented the inhibition of ERK phosphorylation by pressure (Fig. 5A). FAK-Y397 phosphorylation was reduced by pressure in DMSO vehicle control cells and was not affected by SB-203580 pretreatment under ambient pressure. However, the inhibition of FAK-Y397 phosphorylation by pressure was not observed in THP-1 macrophages pretreated with SB-203580 (Fig. 5B). These data raised the possibility that p38 MAPK might influence the downregulation of ERK and FAK activation by pressure.



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Fig. 5. Study of SB-203580 effect on extracellular signal-regulated kinase (ERK) and FAK-Y397 phosphorylation. A: enhancement of ERK phosphorylation by SB-203580. Top: typical Western blot for phosphorylated ERK, stripped and reprobed for total ERK as a loading control, in cells pretreated with DMSO vehicle or SB-203580 under control or increased pressure conditions. Histograph summarizes densitometric results, expressed as means ± SE of the ratio of phosphorylated ERK to total ERK, normalized to DMSO vehicle control. Open bars, ambient pressure; filled bars, increased pressure. Pressure inhibited ERK phosphorylation (*P < 0.05, n = 5) in PMA-differentiated THP-1 macrophages treated with DMSO vehicle control. Pretreatment with SB-203580 enhanced ERK phosphorylation (#P < 0.001, n = 5) and abolished the pressure effect on ERK. B: SB-203580 effect on FAK-Y397 phosphorylation. Top: typical Western blot for phosphorylated FAK-Y397, stripped and reprobed for total FAK as a loading control, in cells pretreated with DMSO vehicle or SB-203580 under control or increased pressure conditions. Histograph summarizes densitometric results expressed as means ± SE of the ratio of phosphorylated FAK-Y397 to total FAK, normalized to DMSO vehicle control. Open bars, ambient pressure; filled bars, increased pressure. Pressure inhibited FAK-Y397 phosphorylation (*P < 0.05, n = 10) in PMA-differentiated THP-1 macrophages treated with DMSO vehicle control. Pretreatment with SB-203580 did not affect FAK-Y397 phosphorylation in ambient pressure condition but abolished the pressure effect on FAK.

 
We therefore next evaluated the effect of siRNA specific for p38 MAPK{alpha} on FAK and ERK expression. In the setting of transfection with p38 MAPK{alpha} to reduce p38 MAPK protein expression, we observed that FAK and ERK protein levels were reduced by 40–50% and by 10–30% in p38 MAPK{alpha} siRNA-transfected THP-1 macrophages, respectively (Fig. 6A). As in the SB-203580 pretreatment experiments, ERK phosphorylation in THP-1 macrophages transfected with p38 MAPK{alpha} increased 1.40 ± 0.10 (P < 0.02, n = 6)-fold under ambient pressure conditions. However, the inhibition of ERK phosphorylation by pressure was prevented in THP-1 macrophages transfected with p38 MAPK{alpha} siRNA (Fig. 6B). In contrast, transfection with p38 MAPK{alpha} siRNA affected neither FAK-Y397 phosphorylation nor the inhibition of FAK-Y397 phosphorylation in response to pressure (Fig. 6C).



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Fig. 6. Effect of p38 MAPK{alpha} siRNA transfection on ERK and FAK expression in PMA-differentiated THP-1 macrophages. A: effect of p38 MAPK{alpha} siRNA on total FAK and ERK expression. Top: typical Western blot for total ERK or FAK, stripped and reprobed for GAPDH as a loading control. Histograph summarizes densitometric analyses, expressed as means ± SE of total ERK or FAK to GAPDH normalized to control scrambled siRNA-transfected cells. Open bars, transfection with scrambled siRNA; hatched bars, transfection with siRNA targeted to p38 MAPK{alpha}. Transfection with siRNA targeted to p38 MAPK{alpha} reduced total p38 MAPK and total FAK (*P < 0.001, n = 4) and total ERK (#P < 0.05, n = 4) expression compared with cells transfected with scrambled siRNA. B: effect of p38 MAPK{alpha} siRNA on ERK phosphorylation. Top: typical Western blot for ERK phosphorylation, stripped and reprobed for total ERK. Histograph summarizes densitometric analyses, expressed as means ± SE of the ratio of phosphorylated ERK to total ERK, normalized to control values in cells transfected with scrambled siRNA and maintained under ambient pressure. Increased extracellular pressure inhibited ERK phosphorylation (*P < 0.02, n = 6, compared with corresponding controls), and transfection with p38 MAPK{alpha} siRNA increased ERK phosphorylation (#P < 0.02, n = 6) and prevented the inhibition of ERK phosphorylation by increased extracellular pressure. C: effect of p38 MAPK{alpha} siRNA on FAK-Y397 phosphorylation. Top: typical Western blot for FAK-Y397 phosphorylation, stripped and reprobed for total FAK. Histograph summarizes densitometric analyses, expressed as means ± SE of the ratio of Y397 phosphorylated FAK to total FAK, normalized to control values in cells transfected with scrambled siRNA. Pressure inhibited FAK-Y397 phosphorylation (*P < 0.02, n = 5, compared with corresponding controls).

 

    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
We have previously reported that a relatively modest increase in extracellular pressure (20 mmHg above ambient) increases serum-opsonized latex bead phagocytosis by PMA-stimulated THP-1 macrophages, at least in part because increased pressure inhibits FAK-Y397 autophosphorylation and consequently inhibits downstream ERK activation (67). The present study demonstrated that extracellular pressure also enhanced phagocytosis by primary isolated human peripheral monocytes and MDM, so this effect is not an artifact of the THP-1 cell line. Inhibition of p38 MAPK activation by SB-203580 or p38 MAPK protein expression by transfection with siRNA targeted to p38 MAPK{alpha} attenuated THP-1 macrophage phagocytosis induced by pressure. Therefore, the effect of pressure on phagocytosis appears to be caused by increased p38 MAPK phosphorylation by pressure. Furthermore, inhibition of p38 MAPK{alpha} increased ERK but not FAK phosphorylation under ambient pressure conditions and blocked the inhibition of ERK activation by pressure.

Normal interstitial tissue pressure in humans is 20–30 mmHg and generally decreases in the setting of acute infection and inflammation (17, 19, 51, 85). Extracellular pressures within inflamed or infected tissues can vary substantially. For example, infection or edema in closed compartments may increase tissue pressure by 5–80 mmHg (8, 64, 65). However, the interstitial or extracellular pressure generally decreases in infected or inflamed tissue lacking such special circumstances (13, 31, 54, 85). Indeed, edema formation in unconstrained inflamed or infected tissues may actually be associated with decreases in connective tissue interstitial fluid pressures by as much as 150 mmHg (85). If the tissue pressure decreases by >10 mmHg or increases over 60 mmHg, pain and cell damage may follow (17, 51). Invasive fungal pathogens such as Pythium insidiosum, a potentially lethal infectious pathogen in humans, exert pressures as high as ~2,000 mmHg (60). Macrophages moving from the capillaries into inflamed tissue are also exposed to capillary pressure elevations. In patients with colonic inflammatory bowel disease, for instance, colonic blood flow is increased two- to sixfold, causing capillary pressure to rise by 10–40 mmHg (18, 24). Activation or inhibition of macrophages by changes in extracellular pressure may therefore affect macrophage function in such settings.

Monocytes and macrophages play a critical role in wound healing, infection, and inflammation (1, 57) and are sensitive to biomechanical stimuli, including cyclic strain, shear, and pressure, although forces of a different nature or magnitude may yield different effects, particularly at different end points (32, 46, 47, 52, 59, 67, 70). Exposure of human MDM to cyclic high pressure (250–1000 mmHg) induces cytokine production (49). We have reported that a constant lower pressure (20 mmHg), which may be more relevant to the cell biology of inflamed tissue, enhances THP-1 macrophage phagocytosis (67), and our current work shows that primary human monocytes behave similarly. Phagocytosis of particles activates several tyrosine kinases that might stimulate further phosphorylation of tyrosine kinases associated with phagosomes in macrophages. However, tyrosine kinases within the cells may also conversely influence the uptake of particles by macrophages.

p38 MAPKs regulate cell growth, cell differentiation, cell activation, and cell survival and respond to inflammation and stress stimuli. In macrophages, p38 MAPKs are activated by cellular stress and microbial phagocytosis (63, 84). However, a role of p38 MAPK in regulating macrophage phagocytosis has not previously been elucidated. Li et al. (41) have reported that p38 MAPK signaling is required for osteoclast differentiation but found that inhibition of p38 MAPK does not affect phagocytosis or cytokine production by murine bone marrow macrophages. Conversely, Hsu et al. (22) have reported that phagocytosis enhanced by polysaccharide from Ganoderma lucidum in neutrophils is mediated by p38 MAPK, phosphatidylinositol 3-kinase, and protein kinase C (22). Mechanical stimuli caused by stretch or pressure have been reported to activate p38 MAPK in some types of cells (2, 4, 44, 75, 76, 80). Our current data suggest that increased p38 MAPK activation in response to extracellular pressure leads to pressure-induced phagocytosis by THP-1 macrophages as well as in primary isolated human peripheral monocytes.

The p38 MAPK family includes four distinct isoforms [p38 MAPK{alpha} (SAPK2a; see Ref. 37), p38 MAPK{beta} (SAPK2b; see Refs. 28 and 71), p38 MAPK{gamma} (SAPK3/ERK6; see Refs. 36 and 42), and p38 MAPK{delta} (SAPK4; see Refs. 29 and 82)] with distinct cellular distribution patterns in mammalian cells. p38 MAPK{alpha} and p38 MAPK{beta} are widely expressed in various cell types and tissues, whereas expression of p38 MAPK{gamma} and p38 MAPK{delta} is more restricted (20, 36, 42). Monocytes express predominantly p38 MAPK{alpha} protein. p38 MAPK{beta} mRNA has been identified in monocytes and macrophages without detectable levels of p38 MAPK{beta} protein (20). Monocytes do not express p38 MAPK{gamma} or p38 MAPK{delta}. After differentiation to macrophages, these cells begin to express p38 MAPK{delta} (20). PMA-differentiated THP-1 macrophages express p38 MAPK{alpha} and p38 MAPK{delta}. In the current study, we chose the p38 MAPK{alpha} isoform as the target gene for siRNA, because the pressure effect was abolished by SB-203580, which inhibits p38 MAPK{alpha} and -{beta}, and also because SB-203580 also abrogated pressure-induced phagocytosis in human peripheral monocytes, which predominantly express p38 MAPK{alpha} and undetectable levels of p38 MAPK{beta} protein and do not express the p38 MAPK{gamma} or -{delta} isoforms. Our results suggest that p38 MAPK{alpha} activation mediates THP-1 macrophage and human peripheral monocyte phagocytosis induced by extracellular pressure. This conclusion is supported by our observations that pressure-induced phagocytosis was prevented by pretreatment with SB-203580 or by transfection with p38 MAPK{alpha} siRNA (21). Furthermore, the augmentation of p38 MAPK phosphorylation by pressure in THP-1 cells transfected with p38 MAPK{alpha} siRNA was attenuated. These data suggest that p38 MAPK activation by pressure might mediate pressure-induced phagocytosis by THP-1 macrophages and human peripheral monocytes.

ERK, another MAPK, is activated by cross-linking of integrins and phagocytosis of particle antigens in macrophages, and ERK activation subsequently influences cytokine expression, cell survival, and control of intracellular pathogen growth. ERK is typically activated by mechanical forces in such other types of cells as osteoblasts (27), smooth muscle cells (34, 75), skeletal muscle fibers (33), intestinal epithelial cells (40, 77), cardiac fibroblasts (81), and cardiac myocytes (45). However, ERK is not activated by pressure in malignant colonocytes in suspension (73) or in rat neonatal cardiac fibroblasts expressing prominent stress fibers and high basal levels of smooth muscle actin (81). Indeed, we previously reported that ERK is actually inhibited in response to increased extracellular pressure in THP-1 cells, together with enhanced phagocytosis (67). ERK blockade mimicked the pressure effect on phagocytosis and substantially attenuated the further stimulation of phagocytosis by pressure.

Cross talk between p38 MAPK and ERK may be important for regulation of cell function, although this has not previously been addressed in human macrophages. We therefore performed experiments to analyze the potential relationship between p38 MAPK and ERK in our system. We found that inhibition of ERK by PD-98059 did not affect basal p38 MAPK phosphorylation or p38 MAPK phosphorylation in response to pressure. This contrasts with the report by Xiao et al. (87), who demonstrated that LPS-induced p38 MAPK phosphorylation is enhanced by ERK inhibition by PD-98059 in the murine macrophage RAW cell line. Xiao et al. did not assess the effects of PD-98059 on basal p38 phosphorylation, however. Taken together, our results and those of Xiao et al. suggest that pressure may modulate p38 phosphorylation by a different pathway than LPS (87).

We also observed that inhibition of p38 MAPK by SB-203580 or by p38 MAPK{alpha} siRNA significantly increased basal ERK phosphorylation in THP-1 macrophages. This is consistent with the previous report by Xiao et al. (87). Furthermore, we also found that p38 MAPK inhibition abolished the inhibition of ERK activation by pressure. In contrast, inhibition of ERK by PD-98050 affected neither basal p38 MAPK activation nor p38 MAPK activation by pressure in THP-1 macrophages. These results suggest that activation of p38 MAPK by pressure may lead to subsequent downstream inhibition of ERK activation.

FAK is a multifunctional tyrosine kinase associated with signaling triggered by integrin cross-linking and expressed by a variety of cell types. FAK protein expression in THP-1 cells is induced after stimulation with PMA and differentiated into macrophage-like cells (67). Activation of FAK by mechanical forces such as pressure has been reported in other types of cells, including colonic cancer cells (40, 73), NIH 3T3 fibroblasts (78), and cardiomyocytes (62, 86), often in parallel with p38 MAPK activation (4, 23, 40, 80). In our system, however, pressure increased p38 MAPK activation but inhibited activation of FAK-Y397 in PMA-differentiated THP-1 macrophages, leading to enhanced latex bead uptake. Inhibition of FAK by transfection with siRNA targeted to FAK also increased THP-1 macrophage phagocytosis.

The relationship between FAK and p38 MAPK in macrophages is poorly understood. Our group has previously shown that a higher amount of activated p38 MAPK in nonadherent SW620 colon cancer cells is associated with decreased FAK-Y397 phosphorylation, suggesting that the cross talk between FAK and p38 MAPK might regulate colon cancer cell adhesion and cell survival (80). Aikawa et al. (4) have reported that inhibition of FAK activation by phosphatase and tensin homolog deleted on chromosome 10 suppresses mechanical stretch-induced p38 MAPK activation in cardiac myocytes and that FAK activation might control p38 MAPK activation. In our current study, we observed that blocking p38 MAPK activity by SB-203580 prevented the inhibition of FAK-Y397 phosphorylation by pressure. On the other hand, pressure-induced inhibition of FAK-Y397 phosphorylation was not affected when p38 MAPK was inhibited by transfection with siRNA targeted to p38 MAPK{alpha}. These differences in p38 MAPK inhibition by SB-203580 treatment and by p38 MAPK{alpha} siRNA transfection may be because of differences in the specificity of the experimental systems. siRNA targeting to p38 MAPK{alpha} specifically silences p38 MAPK{alpha} gene expression and would be expected to exhibit higher specificity than the chemical inhibitor. Although SB-203580 is widely employed as a specific p38 MAPK inhibitor, several studies have reported that SB-203580 possessed some other biological activity besides inhibiting p38 MAPK activity. These have included influencing Raf activation, activation of ERK, inhibition of JNK, and blocking Akt phosphorylation (9, 12, 15, 35, 90). Such nonspecific biological activities might influence pressure-associated changes in FAK-Y397 phosphorylation independent of pressure activation of macrophage p38.

Furthermore, basal p38 MAPK activation in THP-1 macrophages transfected with FAK siRNA did not differ from p38 MAPK activation in cells transfected with scrambled siRNA. However, the enhancement of p38 MAPK activation by pressure was attenuated in THP-1 macrophages transfected with FAK siRNA. These data suggest that the cross talk between p38 MAPK activation and inhibition of FAK-Y397 phosphorylation by pressure might regulate downstream ERK activation, leading to macrophage phagocytosis.

Taken together with our previous work (67), these data suggest that decreased FAK and ERK activity and increased p38 MAPK{alpha} activity are all involved in the mediation of pressure-stimulated phagocytosis. We previously demonstrated that FAK appeared upstream of ERK in this pathway. The present results suggest that FAK inhibition by pressure is also upstream of p38 MAPK{alpha} stimulation by pressure and that p38 activity by pressure is upstream of ERK inhibition by pressure. Whether the FAK effect on ERK occurs solely via p38 MAPK modulation or whether FAK also affects ERK by a p38 MAPK-independent pathway awaits further investigation.

Although these investigations have begun to trace a pattern of intracellular signals by which macrophages may respond to increased extracellular pressure, the mechanism by which this change in extracellular pressure is initially sensed by the macrophage is not yet known. Extrapolation from studies and hypotheses in other cell types suggests that different cell types may sense various physical forces by several competing or complementary mechanisms. These have included tensegrity-type mechanotransduction by the cytoskeleton itself (25, 26), alterations in the activity of membrane ion channels (6, 16, 88), activation of GTP-binding proteins (30), and effects at the level of integrin-mediated cell-matrix interactions (89). Which one or more of these mechanisms may contribute to macrophage pressure sensing awaits elucidation.

There are clearly many differences between macrophage phagocytosis in vitro and immune surveillance in vivo, and p38 modulation also has the potential to affect other aspects of human biology and pathobiology. However, RWJ-67657, BIRB 796 BS, and SB-242235 are experimental drugs that have been used to inhibit p38 MAPK activity in preclinical studies and in an animal model of rat and monkey (10, 11, 56, 83). In the future, it is possible that drugs such as these might be useful to modulate p38 MAPK activity in macrophages to promote phagocytosis, but this possibility awaits substantial further study as well as the elucidation of possible side effects.

In summary, these results demonstrate that constant 20 mmHg pressure increases phagocytosis by primary isolated human monocytes and MDM, as well as in THP-1 macrophages, and that p38 MAPK{alpha} activation by pressure contributes to the enhancement of monocyte/macrophage phagocytosis. Furthermore, cross talk between activation of p38 MAPK{alpha} and inhibition of FAK-Y397 phosphorylation by pressure may modulate the pressure-induced phagocytosis in THP-1 macrophages and human primary monocytes by regulating ERK phosphorylation.


    ACKNOWLEDGMENTS
 
This work was supported in part by National Institute of Diabetes and Digestive and Kidney Diseases Grant R01 DK-60771 (to M. D. Basson).


    FOOTNOTES
 

Address for reprint requests and other correspondence: M. D. Basson, John D. Dingell VA Medical Center, 4646 John R. St., Detroit, MI 48201-1932 (E-mail: marc.basson{at}med.va.gov)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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