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Am J Physiol Cell Physiol 290: C1633-C1639, 2006. First published January 25, 2006; doi:10.1152/ajpcell.00576.2005
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RECEPTORS AND SIGNAL TRANSDUCTION

G protein-coupled receptors serve as mechanosensors for fluid shear stress in neutrophils

Ayako Makino,1 Eric R. Prossnitz,2 Moritz Bünemann,3 Ji Ming Wang,4 Weijuan Yao,1 and Geert W. Schmid-Schönbein1

1Department of Bioengineering, The Whitaker Institute of Biomedical Engineering, University of California, San Diego, La Jolla, California; 2Department of Cell Biology and Physiology, University of New Mexico Health Sciences Center, Albuquerque, New Mexico; 3Department of Pharmacology and Toxicology, University of Würzburg, Würzburg, Germany; 4Laboratories of Molecular Immunoregulation, Center for Cancer Research, National Cancer Institute, Frederick, Maryland

Submitted 17 November 2005 ; accepted in final form 19 January 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Many cells respond to fluid shear stress but in a cell type-specific fashion. Fluid shear stress applied to leukocytes serves to control pseudopod formation, migration, and other functions. Specifically, fresh neutrophils or neutrophilic leukocytes derived from differentiated HL60 cells respond to fluid shear stress by cytoplasmic pseudopod retraction. The membrane elements that sense fluid shear and induce such a specific response are still unknown, however. We hypothesized that membrane receptors may serve as fluid shear sensors. We found that fluid shear decreased the constitutive activity of G protein-coupled receptors (GPCRs). Inhibition of GPCR constitutive activity by inverse agonists abolished fluid shear stress-induced cell area reduction. Among the GPCRs in neutrophils, the formyl peptide receptor (FPR) exhibits relatively high constitutive activity. Undifferentiated HL60 cells that lacked FPR formed few pseudopods and showed no detectable response to fluid shear stress, whereas expression of FPR in undifferentiated HL60 cells caused pseudopod projection and robust pseudopod retraction during fluid shear. FPR small interfering RNA-transfected differentiated HL60 cells exhibited no response to fluid shear stress. These results suggest that GPCRs serve as mechanosensors for fluid shear stress in neutrophils by decreasing its constitutive activity and reducing pseudopod projection.

leukocyte; constitutive activity; mechanotransduction; formyl peptide receptor

THE INFLAMMATORY CASCADE is the basis for tissue repair and wound healing. Increasing evidence, however, shows that subclinical chronic inflammation is an important pathogenic mechanism leading to the development of cardiovascular disease. One of the key events in inflammation is neutrophil activation with pseudopod projection, oxygen free radical production, expression of membrane adhesion molecules, and degranulation. Pseudopod projection per se causes significant flow reduction in the microcirculation and stasis in capillaries, whereas retraction of pseudopods reduces microvascular entrapment and inflammation (16, 34, 38). The state of activation of circulating leukocytes can be controlled by a number of mechanisms traditionally thought of mostly in terms of inflammatory mediators. We demonstrated that fluid shear stress without inflammatory mediators also regulates leukocyte activation, such as pseudopod formation and microcirculatory blood flow (29). In the static state without fluid shear stress, neutrophils have the ability to form pseudopods after contact with different types of surfaces, including coverslips or vessel walls, and they are constitutively activated. Fluid shear controls spontaneous activity of the Rho family small GTPase, Rac, during pseudopod retraction. Deactivation of Rac by fluid shear plays a critical role in leukocyte circulation without entrapment in the capillary network (28). However, the sensor elements in the cell membrane that link extracellular fluid shear stress to intracellular signaling cascades are currently unknown.

Fluid shear controls the morphology of neutrophils differently from, for example, endothelial cells that elongate in the direction of fluid shear or smooth muscle cells that elongate perpendicularly to shear direction (8, 22, 40). The specificity of the response to fluid shear among different cell types suggests that specific mediators on the cell membrane are involved in fluid shear sensing and transduction mechanisms. Thus we hypothesized that G protein-coupled receptors (GPCRs) serve as mechanosensors that respond to fluid shear stress in neutrophils. GPCRs in the plasma membrane exhibit constitutive activity, which is the ability to assume an active conformation in the absence of agonist, sufficient to promote intracellular stimulation of downstream effectors (36). GPCR constitutive activity in the leukocyte plays a critical role in its pseudopod formation. Neutrophils express several Gi protein-coupled chemoattractant receptors, including formyl peptide receptor (FPR), and activated receptors play an important role in cell migration via Rho family small GTPases (1, 32, 41). Because FPR exhibits relatively high constitutive activity among other chemoattractant receptors and is expressed predominantly in neutrophils and monocytes but less in other cell types (21), FPR may be a mechanosensor candidate for leukocytes.

In this study, we have used human promyeloid HL60 cells differentiated by DMSO into neutrophils to facilitate the use of transfection methods and allow the expression of exogenous proteins (28, 33). HL60 cells exhibit the ability to differentiate into morphologically mature myeloid cells with many of the markers and capabilities of neutrophils (9, 17, 33). Differentiated HL60 cells have been used extensively as a model system for studying neutrophil function, and they exhibit a robust fluid shear response (28).

We report herein the role of GPCRs as mechanosensors in fluid shear stress-induced pseudopod retraction in neutrophils. Fluid shear may control pseudopod formation via a decrease in GPCR constitutive activity and thereby maintain stable circulation of leukocytes.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Biological materials and reagents. Human promyelocytic leukemia HL60 cells [American Type Culture Collection (ATCC), Manassas, VA] were cultured in RPMI 1640 (Invitrogen, Carlsbad, CA) supplemented with 10% heat-inactivated FBS (Mediatech, Herndon, VA) in 5% CO2 at 37°C. HL60 cell differentiation was induced by treatment with 1.4% DMSO (ATCC) for 5 days. The mouse anti-FPR MAb was obtained from BD Pharmingen (San Diego, CA). HBSS and N-formyl-Nleu-Leu-Phe-Nleu-Tyr-Lys fluorescein derivative were obtained from Invitrogen. The protease inhibitors, PMSF, aprotinin, and leupeptin, the chemoattractant, formyl-methionyl-leucyl-phenylalanine (fMLP), pertussis toxin (PTX), and monensin were purchased from Sigma Chemical (St. Louis, MO).

Fluid shear application in a flow chamber. Flow chamber studies were performed as previously described (28). Cells were suspended in RPMI 1640 containing 10% FBS and loaded into the flow chamber. Laminar shear stress (5 dyn/cm2) was generated by perfusion of HBSS (pH 7.4 adjusted with 10 mM HEPES containing Ca2+ and Mg2+ at 1 mM each) with an automated syringe pump (Harvard Apparatus, Holliston, MA) attached to the inlet side of the flow chamber. The cell responses that we observed in the present study were all evoked by physiological but comparatively small fluid shear stress levels (5 dyn/cm2, or ~5/981 cmH2O) that were far below the values required to achieve a passive viscoelastic response (~100–1,000 dyn/cm2) (42). Images of the cells were recorded using a x60 magnification lens objective and a 1.9 numerical aperture on a charge-coupled device camera (model VI-470; Optronics, Goleta, CA) and stored digitally (Scion Image software; Scion, Frederick, MD). Images were recorded during 10 min of fluid shear stress and for 10 min during the recovery phase. While neutrophils adhere to a coverslip, they project cytoplasmic regions designated as pseudopods, regardless of their particular shapes, such as veillike lamellipodia, fingerlike filopodia, and uropodia at the tailing edge. Each cell contour was outlined manually, and the area within the outlined shape was computed (Scion Image software). This measurement is referred to as projected cell area.

Transient transfection of Gi proteins. Gi protein construction was prepared as previously described (5). Enhanced yellow fluorescent protein (YFP) (F46L) was inserted into the A-B loop within the {alpha}-helical domain of the Gi{alpha}1 subunit. Gbeta1{gamma}2 subunits were fused to cyan fluorescent protein (CFP) on the NH2 terminus of Gbeta1 (CFP-N-Gbeta1). A heterotrimeric formation of Gi{alpha}1-YFP, CFP-N-Gbeta1, and G{gamma}2 can cause an increase in the fluorescence resonance energy transfer (FRET)-to-CFP ratio. HL60 cells were cultured in the presence of 1.4% DMSO for 4 days to initiate differentiation before transfection. On day 4, the cells were washed and suspended in Opti-MEM reduced serum medium at 2 x 106 cells/well. The cells were transfected with cDNA using the cationic lipid DMRIE-C transfection reagent (1,2-dimyristyloxypropyl-3-dimethyl-hydroxy ethyl ammonium bromide; Invitrogen). cDNA (4.9 µg; 3.6 µg of Gi{alpha}1-YFP, 0.5 µg of CFP-N-Gbeta1, and 0.8 µg of G{gamma}2) were suspended in Opti-MEM and mixed with the cationic lipid DMRIE-C (4.9 µl/well). In pilot studies, we used different ratios of Gi{alpha}1-YFP, CFP-N-Gbeta1, and G{gamma}2 according to the original cDNA concentrations used for other cell types (5) and selected a 3.6-to-0.5-to-0.8 ratio for the experiments. This ratio provided a better FRET signal after stimulation than any other cDNA concentration ratio in the cell line used in this study. The cells were suspended in the cationic lipid-cDNA mixture and incubated at 37°C for 5 h. An equal volume of RPMI 1640 medium containing 20% heat-inactivated FBS plus DMSO (1.4% final concentration) was then added, and the cells were cultured for 20 h. On day 5, the cells were washed and suspended in fresh RPMI containing 10% FBS and cultured for an additional 24 h before the experiment.

FRET imaging. For FRET imaging, we used an inverted microscope equipped with a x60 magnification oil-immersion lens objective, a cooled charge-coupled device camera, a filter changer, a 440DF20 excitation filter, a 455DRLP diachroic mirror, and two emission filters (535DF25 and 480DF30). Illumination time was set to 300–400 ms, and images were captured once per minute during the experiment and digitally analyzed (Simple PCI software, version 5.2; Compix Imaging System, Cranberry Township, PA). The FRET signal was determined as the ratio of the YFP-to-CFP (FRET-to-CFP) emission. A background image was recorded under exactly the same conditions without cells, and the background fluorescence intensity was subtracted.

Stable transfection of HL60 cells. FPR small interfering RNA (siRNA) or random RNA sequence (mock) were constructed into the pSIREN-RetroQ retroviral vector (Clontech Laboratories, Mountain View, CA) (20). HL60 cells were transfected using electroporation (gene pulser; Bio-Rad Laboratories, Hercules, CA). Cells (2 x 106) were centrifuged and resuspended in 400 µl of Opti-MEM reduced serum medium (Invitrogen). Vector (10 µg) was added to the cells and preincubated for 5 min on ice. The cells were then subjected to a single 200-V pulse from a 960-µF capacitor and returned to 10 ml of culture medium. The next day, 1 µg/ml puromycin was added to the medium. As the selection proceeded, the cells were centrifuged and resuspended in fresh medium (containing puromycin) at 3- to 5-day intervals. As the cell density began to increase between 13 and 16 wk posttransfection, the medium was changed as needed to maintain cell density at <2 x 106 cells/ml. FPR cDNA was transfected as described by Prossnitz et al. (33).

Ligand binding assay with flow cytometry. The cells were harvested by centrifugation and resuspended in cold HBSS (106 cells/ml). Binding was conducted with N-formyl-Nleu-Leu-Phe-Nleu-Tyr-Lys fluorescein derivative at 10 nM concentration. After incubation for 20 min on ice, ligand binding was analyzed using flow cytometry (Becton Dickinson, San Jose, CA). Debris and dead cells were excluded by gating forward and side scatter. Nonspecific binding was determined in the presence of 1 M fMLP.

RNA isolation and RT-PCR. RT-PCR was performed as described previously by Le et al. (20). DNA-free total RNA was extracted from cells (RNeasy Mini kit; Qiagen, Valencia, CA). Total RNA (0.1 µg) was reverse transcribed (iScript cDNA synthesis kit; Bio-Rad Laboratories). PCR (GeneAmp PCR system; PerkinElmer, Boston, MA) was performed using AccuPrime Taq DNA Polymerase (Invitrogen). The sense and antisense primers were specially designed from the coding regions of FPR genes (20). beta-Actin was used as a control. PCR products were visualized using ethidium bromide staining in 1.2% agarose gel.

Statistical analysis. Data are presented as means ± SE. For comparison of groups, two-way repeated-measures ANOVA was performed and the Bonferroni-Dunn test was used for post hoc analysis. Comparisons between groups of maximum responses (%) were conducted using an unpaired Student's t-test as summarized in Table 1.


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  		Table 1. Summary of cell response to fluid shear

 

   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
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Fluid shear decreases Gi protein constitutive activity in differentiated HL60 cells. The FRET technique was used to visualize Gi protein activity. Gi{alpha}1-YFP, CFP-N-Gbeta1, and G{gamma}2 were cotransfected transiently in the differentiated HL60 cells, and the ratio of FRET to CFP was measured during the application of fluid shear stress. With this construction, the binding of the G{alpha} subunit to the Gbeta{gamma} subunit causes a FRET signal (5). As a control experiment for differentiated HL60 cells transfected with Gi proteins, FPR was stimulated using fMLP (10–8 M), which led to a significantly increased FRET ratio (control 1.349 ± 0.025 vs. fMLP 1.708 ± 0.018; *P < 0.05) (Fig. 1A), suggesting that the Gi activity level was increased by coupling of FPR to G{alpha}i1. Before fluid shear application, the cells exhibited some degree of FRET signal at the basal level, indicating that the differentiated HL60 cells were constitutively active during pseudopod projection. Physiological fluid shear significantly and rapidly decreased the constitutive activity of GPCRs (Fig. 1B).


Figure 1
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  		Fig. 1. Fluid shear decreases Gi protein constitutive activity in differentiated HL60 cells. A: control experiment. Fluorescence photomicrographs of differentiated HL60 cells transfected with Gi proteins show fluorescence resonance energy transfer (FRET) and cyan fluorescent protein (CFP) signal. The Gi protein activity is displayed in pseudocolor before and after stimulation with formyl-methionyl-leucyl-phenylalanine (fMLP; 10–8 M). An increase in the FRET-to-CFP ratio indicates activation of Gi protein. Graph below photomicrographs show normalized FRET ratios in differentiated HL60 cells (light gray bar, control; dark gray column, fMLP; n > 30 cells in each group). *P < 0.05 vs. ratio of control cells. Error bars, SE. B: Gi protein activity detected using FRET in differentiated HL60 cells during fluid shear. Cells were subjected to fluid shear for 10 min in the flow chamber. Top: pseudocolor images of Gi protein activity before and after fluid shear; bottom: normalized FRET ratio in differentiated HL60 cells during shear application (n = 8). *P < 0.05 vs. ratio during static condition (before shear application). Error bars, SE.

 
Fluid shear-induced reduction of cell spreading is associated with deactivation of GPCR constitutive activity. Fluid shear caused rapid and significant rounding of the differentiated HL60 cells as detected by reduction of the projected cell area (28). To examine the effects of inverse agonist of GPCR constitutive activity on fluid shear-induced cell response, differentiated HL60 cells were treated overnight with 20 ng/ml PTX in culture medium and then subjected to fluid shear stress in the flow chamber. In the presence of PTX, the cells not only responded less to fluid shear but also demonstrated a marked increase in the degree of cell spreading during the last 4 min of shear stress exposure (Fig. 2A). The rise in cytosolic Na+ concentration ([Na+]cyt) by pretreatment with monensin, a Na+ ionophore, served to block the fluid shear stress-induced reduction in cell spreading (Fig. 2B), suggesting that fluid shear attenuates cell spreading via control of GPCR constitutive activity.


Figure 2
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  		Fig. 2. Fluid shear-induced reduction of cell spreading is associated with deactivation of G protein-coupled receptor (GPCR) constitutive activity. A: 20 ng/ml pertussis toxin (PTX) overnight treatment (bullet; n = 10) and control ({circ}; n = 8). B: 10–6 M monensin (bullet; n = 8) and 0.01% ethanol as a control ({circ}; n = 7). Error bars, SE. For statistics, see Table 1.

 
Response to fluid shear in undifferentiated HL60 cells. Undifferentiated HL60 cells do not express chemoattractant receptors in the plasma membrane, including the FPR (33). After differentiation, HL60 cells strongly express FPR mRNA and FPR in the plasma membrane (Fig. 3, A and B). Before application of fluid shear stress, differentiated HL60 cells had been migrating on the coverslip and spreading their cytoplasm, and fluid shear stress induced a rapid and significant reduction of projected cell area. In contrast, undifferentiated HL60 cells had a low ability to form pseudopods while adhering to the wall of the flow chamber and demonstrated no detectable response to fluid shear stress (Fig. 3C).


Figure 3
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  		Fig. 3. Response of undifferentiated and differentiated HL60 cells to fluid shear. A: representative examples and summary measurements of formyl peptide receptor (FPR) mRNA expression in undifferentiated HL60 cells (HL) and differentiated HL60 cells (DHL) (n = 2 for each). B: expression of surface membrane FPR in HL and DHL. Dotted line indicates nonspecific binding (Non). C: projected cell area of HL ({circ}; n = 10) and DHL (bullet; n = 8) during fluid shear stress at 5 dyn/cm2 for 10 min in the flow chamber. Photomicrographs show each cell before and after application of fluid shear stress. Bar, 10 µm. Error bars, SE. For statistics, see Table 1.

 
FPR transfection in HL60 cells induces pseudopod formation and restores the cell response to fluid shear. To examine whether fluid shear stress causes cell retraction via FPR, we transfected the FPR cDNA in undifferentiated HL60 cells using electroporation (33). cDNA encoding only green fluorescent protein (GFP) was transfected into the cells as a control. FPR cDNA transfection enhanced the expression of FPR protein on the surface membrane in undifferentiated HL60 cells, which, without FPR cDNA transfection, showed low levels of FPR. The expression levels starting from the lowest levels were: undifferentiated HL60 cells < differentiated HL60 cells < FPR-transfected undifferentiated HL60 cells < FPR-transfected differentiated HL60 cells (Fig. 4A).


Figure 4
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  		Fig. 4. FPR transfection in undifferentiated HL60 cells induces pseudopod formation and restores fluid shear response. A: expression of surface membrane FPR in undifferentiated HL60 cells, differentiated HL60 cells, FPR-transfected undifferentiated HL60 cells (HLFPR), and FPR-transfected differentiated HL60 cells (DHLFPR). B: typical image of GFP-transfected undifferentiated HL60 cells (HLGFP) and FPR-transfected undifferentiated HL60 cells (HLFPR) during migrating on the cover slip. C and D: projected cell area of HLGFP ({circ} in C; n = 5), HLFPR (bullet in C; n = 10), GFP-transfected differentiated HL60 cells (DHLGFP; {circ} in D; n = 6), and DHLFPR (bullet in D; n = 8) during fluid shear application. Error bars, SE. For statistics, see Table 1.

 
Importantly, expression of FPR in undifferentiated HL60 cells causes pseudopod projection (Fig. 4B) and pseudopod retraction during fluid shear (Fig. 4C). Furthermore, overexpression of FPR by differentiation of FPR-transfected HL60 led to even stronger pseudopod retraction during fluid shear compared with differentiated HL60 cells transfected only with GFP (Fig. 4D).

Inhibition of FPR expression attenuates the fluid shear-induced response. To investigate whether GPCRs other than FPR might facilitate fluid shear-induced reduction of cell spreading, we examined whether FPR siRNA could affect the cellular response to fluid shear. Undifferentiated HL60 cells were stably transfected with FPR siRNA by electroporation. Mock-transfected cells were used as the control. Transfected cells were differentiated and then used for the flow chamber study. FPR siRNA transfection in HL60 cells successfully downregulated the levels of FPR mRNA (Fig. 5A) and surface membrane FPR expression (Fig. 5B) compared with mock-transfected cells. FPR siRNA-transfected differentiated HL60 cells, which expressed other receptors except for FPR, significantly decreased the cell response to fluid shear compared with mock-transfected differentiated HL60 cells (Fig. 5C). These results suggest that FPR plays a central role in pseudopod retraction during fluid shear.


Figure 5
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  		Fig. 5. Inhibition of FPR expression in differentiated HL60 cells attenuates the fluid shear stress-induced response. A: representative examples and summary measurements of FPR mRNA expression (n = 2 for each). B: expression of surface membrane FPR in mock-transfected differentiated HL60 cells (D-Mock) and FPR small interfering RNA (siRNA)-transfected differentiated HL60 cells (D-siRNA). C: projected cell area of D-Mock ({circ}; n = 11), and D-siRNA (bullet; n = 9). Fluid shear stress was 5 dyn/cm2 in the flow chamber. Error bars, SE. For statistics, see Table 1.

 

   DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Altogether, these results indicate that in neutrophils, the fluid shear stress-induced signaling pathway with reduction of cell spreading due to pseudopod formation is initiated by deactivation of the constitutive activity of GPCR. FRET permits the detection of protein-protein interactions in living cells and also enables determination of G protein activity (5). FPR has the ability to couple to all subtypes of Gi{alpha} proteins, including Gi{alpha}1 (36, 39), although it couples mainly to Gi{alpha}2 and Gi{alpha}3 in HL60 cells (14, 31). In this study, we tested Gi{alpha}1 protein activity because Gi{alpha}1 generates much higher FRET signals compared with background, whereas Gi{alpha}2 and Gi{alpha}3 do not generate a sufficient FRET signal-to-noise ratio (12). We also observed a significant increase in the FRET ratio after stimulation with fMLP, an agonist for FPR (Fig. 1A). Fluid shear stress significantly decreased the FRET ratio within seconds in differentiated HL60 cells (Fig. 1B), indicating a reduction in GPCR constitutive activity. In this study, we used differentiated HL60 cells instead of FPR siRNA-transfected differentiated HL60 cells, because we observed enhanced cell apoptosis after cotransfection of Gi proteins into the FPR siRNA-transfected HL60 cells.

The two-state model assumes that GPCRs exist between an inactive state (R) state and an active state (R*) (19, 24, 25, 36). The R-to-R* isomerization of GPCRs during constitutive activity can occur spontaneously, i.e., independently of agonist. Basal constitutive activity of GPCRs can be blocked by inverse agonists but not by receptor antagonists. PTX serves to inhibit constitutive activity of Gi protein-coupled receptors (7, 18, 23), and the monovalent cation Na+ acts as an allosteric inverse agonist that stabilizes the R state and diminishes basal G protein activity (6, 10, 15, 36). Fluid shear stress rapidly reduced cytoplasmic spreading of differentiated HL60 cells. In the presence of PTX, the pseudopod retraction during fluid shear was minimized and cell spreading was observed (Fig. 2A). The reason for this phenomenon may be that the blockade of Gi/o by PTX increased the activities of other types of G proteins (2, 4) and subsequently caused pseudopod formation via fluid shear stress. The rise in cytosolic [Na+]cyt upon monensin treatment diminished the fluid shear-induced reduction in cell spreading (Fig. 2B). These results suggest that in differentiated HL60 cells, fluid shear decreases GPCR constitutive activity together with pseudopod retraction.

Differentiated HL60 cells expressed FPR in the plasma membrane (Fig. 3, A and B), migrated on a coverslip, and spread their cytoplasm before shear stress was initiated. Fluid shear stress induced a rapid and significant reduction of projected cell area (Fig. 3C). This fluid shear stress response is largely missing in undifferentiated HL60 cells because of the absence of surface membrane receptors (e.g., FPR) required to initiate pseudopod projection (33). Expression of only FPR in undifferentiated HL60 cells restored their ability to project pseudopods that they retract under the influence of fluid shear (Fig. 4). In contrast, suppression of only FPR expression in differentiated HL60 cells attenuated their ability to respond to fluid shear stress (Fig. 5). These results thus suggest that the FPR serves as a mechanosensor for fluid shear.

In addition to FPR, two other receptor types in the FPR family have been identified: FPR-like 1 (FPRL1) and FPR-like 2 (FPRL2). FPR and FPRL1, but not FPRL2, are detected in neutrophils. FPR is defined as a high-affinity fMLP receptor (nM range); FPRL1 is characterized as a low-affinity fMLP receptor (µM range); and FPRL2 does not bind or respond to fMLP (21). In this study, we focused on FPR because its constitutive activity has been well characterized (36, 37). Neutrophils have other chemoattractant receptors in the plasma membrane, including complement components C5a and C3a receptors, platelet-activating factor receptor, and leukotriene B4 receptor. These receptors also exhibit constitutive activity (36, 37). FPR-transfected undifferentiated HL60 cells expressed more FPR in the surface membrane than differentiated HL60 cells did (Fig. 4A), but the maximum response induced by fluid shear stress in FPR-transfected undifferentiated HL60 cells (Fig. 4C) was not as strong as that in differentiated HL60 cells (Fig. 3C). Therefore, other receptors may also mediate cell retraction in response to fluid shear stress.

Fluid shear may control FPR constitutive activity by 1) fluid shear induced influx of Na+ and 2) a direct change in the conformation of FPR. A number of other sensory molecules for fluid shear were proposed mainly for vascular endothelium, such as heparan sulfate proteoglycans (11, 30), caveolae (35), flow-sensitive K+ and Cl– channels, integrin, membrane lipids, receptor tyrosine kinases, and GPCRs (3, 26). We have shown that fluid shear cleaves and redistributes the CD18 extracellular domain of neutrophils (13). CD18 also exhibits spontaneous activity and controls cell migration (27). Its exact role in pseudopod retraction remains to be identified.

In summary, our data demonstrate that GPCRs serve as mechanosensors in neutrophils that mediate the retraction of pseudopods under fluid shear stress. Control of GPCR constitutive activity may facilitate stable leukocyte circulation in the bloodstream and may serve as a new target for therapeutic approaches to inflammation to minimize impaired passage of neutrophils with pseudopods through the microcirculation.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by National Heart, Lung, and Blood Institute Program Project Grants HL-43026 and in part by HL-10881.


   ACKNOWLEDGMENTS
 
We thank Dr. Sheng Tong for assistance with FRET imaging analysis, Gerard Norwich for technical assistance with FRET microscopy, and Laura Berstis for assistance with tissue cultures.


   FOOTNOTES
 

Address for reprint requests and other correspondence: G. W. Schmid-Schönbein, Dept. of Bioengineering, The Whitaker Institute of Biomedical Engineering, Univ. of California, San Diego, 9500 Gilman Dr., La Jolla, CA 92093 (e-mail: gwss{at}bioeng.ucsd.edu)

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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