264230 Shear-Induced Resistance to Neutrophil Activation Via the Formyl Peptide Receptor
Shear-Induced Resistance to Neutrophil Activation via the Formyl Peptide Receptor
Michael J. Mitchell1 and Michael R. King1
1Department of Biomedical Engineering, Cornell University, Ithaca, NY 14853 USA
The adhesion of leukocytes to the luminal surface of the microvasculature plays an important role in the inflammatory response and lymphocyte homing to lymphatic tissues (1). The initial step of the leukocyte adhesion cascade involves the capture and rolling of leukocytes on the receptor bearing endothelial cell layer, with L-selectin acting as an important mediator on the leukocyte surface. L-selectin is constitutively expressed on microvilli tips of neutrophils, and is rapidly cleaved from the neutrophil surface due to an inflammatory stimulus. Firm adhesion to the endothelium is mediated via ICAM-1 on endothelial cells binding to β2 integrins on neutrophils. Stimulus by fMLP, which binds to the formyl peptide receptor (FPR) on neutrophils, induces αMβ2 integrin conformational changes and downregulation of L-selectin, which are key indicators of neutrophil activation (2).
FPR, a chemoattractant G-protein coupled receptor (GPCR), exhibits high constitutive activity in addition to activity due to agonist binding (3). Under static conditions, neutrophils spread their cytoplasm and migrate on glass substrates, in part due to constitutive GPCR activity. In the presence of fluid shear stress, neutrophils rapidly retract lamellipodia, assume a round resting state, and decrease GPCR constitutive activity (4). Neutrophils treated with pertussis toxin significantly attenuated the fluid shear stress-induced pseudopod retraction response, demonstrating the role of GPCR activity changes due to fluid shear stress. Transfection of cDNA for FPR into HL60 cells with low levels of FPR and low pseudopod activity led to the projection of pseudopods, which then retracted following exposure to fluid shear stress. FPR depletion via siRNA delivery in differentiated HL60 cells significantly reduced fluid shear stress-induced pseudopod retraction.
While the ability of activated leukocytes to retract pseudopods in response to fluid shear stress has been documented, a variety of different responses have been observed. Leukocytes treated with dexamethasone or after centrifugation have shown to reverse their shear stress response, and can project pseudopods when exposed to fluid shear stress (5). The effect of fluid shear stress on earlier indicators of neutrophil activation, such as L-selectin shedding and αMβ2 integrin activation induced by fMLP, has not yet been addressed. Here, we examined the quantitative dynamics of the shear stress-dependent response of fMLP-induced L-selectin shedding and αMβ2 integrin activation in neutrophils. <>II. Results
<>A. Fluid shear stress reduces fMLP-induced L-selectin shedding and αMβ2 integrin activation in neutrophils
We initially studied the fluid shear stress response of neutrophils to fMLP-induced activation. Neutrophils were exposed to static conditions or 4.0 dyn/cm2 of fluid shear stress in a cone-and-plate viscometer for 2 h at 23°C, followed by stimulation with 0.5 nM fMLP for 10 min. Neutrophils exposed to static conditions (Fig. 1A) or shear stress (Fig. 1D) in the absence of fMLP did not show appreciable differences in activation, as expected. However, neutrophils exposed to fMLP after fluid shear stress (Fig. 1E) showed a measurable reduction in L-selectin shedding and αMβ2 integrin activation compared to neutrophils exposed to static conditions (Fig. 1B) followed by fMLP. A significant reduction in fMLP-induced L-selectin shedding and αMβ2 integrin activation was found in sheared neutrophils compared to neutrophils under static conditions.
Figure 1: Neutrophils exposed to static conditions (A) and 4 dyn/cm2 of shear (B) for 2 h. Neutrophils exposed to static conditions and 4 dyn/cm2 for 2 h then exposed to 0.5 nM fMLP (C, D) or 5 nM IL-8 for 10 min (E, F). Upper two quadrants of plots represent CBRM1/5 staining. Two righthand quadrants represent L-selectin staining. Quadrants determined using isotype antibodies labeling nonspecific binding sites on neutrophils. PE: R-Phycoerythrin. FITC: fluorescein isothiocyanate. <>B. Fluid shear stress does not affect IL-8-induced L-selectin shedding and αMβ2 integrin activation
To assess whether fluid shear stress alters activation via other major GPCRs, neutrophils were exposed to static conditions or fluid shear stress, followed by stimulation with 5 nM of IL-8, which binds to GPCRs CXCR1 and CXCR2. Neutrophils exposed to static conditions (Fig. 1C) or fluid shear stress (Fig. 1F) followed by IL-8 did not show significant differences in L-selectin shedding or αMβ2 integrin activation. <>C. fMLP-induced L-selectin shedding and αMβ2 integrin activation is fluid shear stress dose-dependent
To study the effect of shear stress magnitude on fMLP-induced activation, neutrophils were exposed to shear stresses of 0.1-4.0 dyn/cm2 over 2 h. At shear stresses of 0.10 and 0.25 dyn/cm2, no significant differences in L-selectin shedding (Fig. 2A) or αMβ2 integrin activation (Fig. 2C) were observed between cells exposed to shear and static conditions followed by fMLP. However, a shear stress of 0.75 dyn/cm2 yielded a significant reduction in L-selectin shedding (Fig. 2A) and αMβ2 integrin activation (Fig. 2C) after fMLP exposure. Shear stresses of 2.5 and 4.0 dyn/cm2 showed an even greater reduction of neutrophil activation. <>D. fMLP-induced L-selectin shedding and αMβ2 integrin activation is shear stress exposure time-dependent
To assess the kinetics of the resistance response, neutrophils were exposed to a shear stress of 4.0 dyn/cm2 while increasing exposure time from 1-120 min. No significant difference in fMLP-induced L-selectin shedding in neutrophils was found over a 1-10 min shear stress exposure time (Fig. 2B). A significant decrease in fMLP-induced L-selectin shedding was found at a threshold exposure time of 20 min, and was significantly less than neutrophils in static conditions over a 20-120 min range. fMLP-induced αMβ2 integrin activation in showed no significant difference in neutrophils exposed to shear and static conditions over 1-30 min (Fig. 2D). A threshold value of 60 min was required to produce a significant difference in αMβ2 integrin activation in neutrophils.
Figure 2: Increasing shear stress reduces fMLP-induced L-selectin shedding (A) and aMβ2 integrin activation (C). Shear stress varied in experiments from 0.1-4.0 dyn/cm2 for 120 min, followed by 0.5 nM fMLP stimulation. Time dependence of resistance to fMLP-induced L-selectin shedding (B) and aMβ2 integrin activation (D) determined by increasing exposure time from 1-120 min at 4.0 dyn/cm2. n = 5 donors. *P < 0.05. <>E. Fluid shear stress reduces FPR surface expression
To investigate shear stress effects on FPR surface expression, neutrophils were exposed to shear stress and static conditions and immediately labeled with anti-FPR antibodies for flow cytometry analysis. Sheared neutrophils displayed a reduction in FPR expression (Fig. 3A) compared to nonsheared samples. A significant difference in FPR receptor count was revealed, as sheared neutrophils averaged 14,600 receptors/cell, while nonsheared samples averaged 20,100 receptors/cell (Fig. 3B). IL-8 receptor CXCR1 and CXCR2 receptor counts showed no significant differences due to fluid shear stress (Fig. 3A,B).
Figure 3: Flow cytometry plots show decreased FPR expression at 4.0 dyn/cm2 for 2 h (A) compared to nonsheared samples. Surface receptor counts of FPR, CXCR1, and CXCR2 on neutrophils (B). FPR internalization measured via image thresholding (C) to exclude cell membrane from measurements. FPR internalization when exposed to 4.0 dyn/cm2 (D) of fluid shear stress compared to those exposed to static conditions (E). Fluorescent intensity of the intracellular region (F) was quantified. Scale bars = 5 µm. n = 3 donors. *P < 0.05. <>F. Neutrophils undergo FPR internalization under fluid shear
To examine FPR internalization as a cause for surface expression decrease following shear stress exposure, neutrophils were exposed to fluid shear stress or static conditions for 2 h, permeabilized and labeled with FPR antibodies for examination via confocal microscopy. Images were thresholded (Fig. 3C) to exclude the cell membrane from fluorescence measurements. Immunostaining revealed FPR to be clearly localized within the cell in sheared samples (Fig. 3D), while minimal FPR was shown within neutrophils exposed to static conditions (Fig. 3E). Average pixel intensities of the intracellular region showed a significant increase in fluorescence intensity in sheared neutrophils, compared to neutrophils exposed to static conditions (Fig. 3F).
Results from this study suggest that fluid shear stress has a significant effect on the activation of circulating neutrophils. Neutrophils acquired a fluid shear stress-induced resistance to activation via FPR. Resistance was shown to be dependent on shear stress magnitude, as the response increased with increasing shear stress. The mechanical response was also dependent on shear stress duration, as neutrophils increased resistance with increased shear exposure time. A decrease in FPR surface expression was observed upon exposure shear stress, and high-resolution confocal microscopy revealed FPR internalized within cells. While other studies on mechanotransduction in neutrophils mostly focused on morphological changes, the present study focused on earlier indicators of activation, specifically fMLP-induced L-selectin shedding and αMβ2 integrin activation. The complete signaling pathways of these receptors deserve further study, along with molecules that mediate GPCR internalization. Other receptors that display high constitutive activity should be investigated to understand their contributions to the mechanosensing response of cells within the vascular microenvironment. <>References
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