(74f) 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 β₂integrins
on neutrophils. Stimulus by fMLP, which binds to the formyl peptide receptor (FPR) on neutrophils, induces α_Mβ₂
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 α_Mβ₂ 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 α_Mβ₂ integrin activation in neutrophils.
II.    Results

A.   
Fluid shear stress reduces fMLP-induced
L-selectin shedding and α_Mβ₂
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 α_Mβ₂ integrin
activation compared to neutrophils exposed to static conditions (Fig. 1B)
followed by fMLP. A significant reduction in fMLP-induced L-selectin shedding
and α_Mβ₂ 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 α_Mβ₂ 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 α_Mβ₂
integrin activation.
C.   
fMLP-induced
L-selectin shedding and α_Mβ₂
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 α_Mβ₂ 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 α_Mβ₂ 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 α_Mβ₂
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
α_Mβ₂ 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 α_Mβ₂ integrin
activation in neutrophils.

Figure 2:
Increasing shear stress reduces fMLP-induced L-selectin shedding (A)
and a_Mβ₂
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 a_Mβ₂
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).

III.    Conclusion

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 α_Mβ₂
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.
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