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Originally published online as doi:10.1189/jlb.0507304 on October 10, 2007

Published online before print October 10, 2007
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(Journal of Leukocyte Biology. 2008;83:99-105.)
© 2008 by Society for Leukocyte Biology

Neutrophil adhesion to E-selectin under shear promotes the redistribution and co-clustering of ADAM17 and its proteolytic substrate L-selectin

Ulrich Schaff*,1, Polly E. Mattila{dagger},1, Scott I. Simon* and Bruce Walcheck{dagger},2

{dagger} Department of Veterinary and Biomedical Sciences, University of Minnesota, St. Paul, Minnesota, USA; and
* Department of Biomedical Engineering, University of California, Davis, California, USA

2 Correspondence: University of Minnesota, 295j AS/VM Bldg., 1988 Fitch Avenue, St. Paul, MN 55108, USA. E-mail: walch003{at}umn.edu


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ABSTRACT
 
E-selectin is expressed by the vascular endothelium and binds flowing neutrophils in the blood to facilitate their recruitment into the underlying tissue at sites of inflammation. L-selectin on neutrophils is engaged by E-selectin and undergoes rapid clustering and then coalescence in the trailing edge of polarizing cells. These processes are believed to increase the valency and capacity of L-selectin to signal CD18 integrin activity. Neutrophils, upon exiting the microvasculature, down-regulate their surface L-selectin through ectodomain shedding by a disintegrin and metalloprotease 17 (ADAM17). We reasoned that neutrophil tethering and rolling on E-selectin might initiate a coordinate change in the membrane distribution of ADAM17 as well. We found that ADAM17 indeed underwent a dramatic cell surface redistribution to the trailing edge of neutrophils rolling on purified E-selectin when activated by a chemoattractant under shear flow; however, its lateral migration occurred at a slower rate than L-selectin. ADAM17 and L-selectin also redistributed in the same manner in neutrophils attached to IL-1β-stimulated HUVEC under shear flow. In contrast, the coalescence of L-selectin on the surface of neutrophils by antibody cross-linking did not promote the redistribution of ADAM17, suggesting that these molecules do not constitutively associate in the plasma membrane. Together, our findings reveal that neutrophil activation upon E-selectin adhesion initiates active transport of ADAM17 and L-selectin to the cell uropod, thus providing additional insight into the molecular mechanisms that regulate L-selectin during leukocyte extravasation.

Key Words: ectodomain shedding • inflammation


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INTRODUCTION
 
Neutrophils are the most prominent leukocyte component of blood and at sites of acute inflammation following their emigration through the microvasculature. The adhesion molecule L-selectin (CD62 ligand) is constitutively expressed on neutrophils and is critical for directing these cells to diverse sites of inflammation [1 ]. Sialyl Lewisx on L-selectin is recognized by the vascular adhesion molecule E-selectin [2 ]. Current data reveal that neutrophil interactions with E-selectin result in a dramatic shift in the membrane topography of L-selectin, which undergoes a rapid redistribution from its uniform membrane dispersal to punctate clusters and eventually congregates at the trailing edge (uropod) of neutrophils adhered to purified E-selectin and cytokine-inflamed endothelium [3 , 4 ]. The lateral movement of L-selectin involves an active transport process mediated by its association with the actin cytoskeleton and membrane microdomains [5 ]. The formation of L-selectin clusters may function to increase the valency of interactions with counter-receptors. Mathematical simulations of the impact of L-selectin clustering on neutrophil rolling on E-selectin predict periodic occurrences of low rolling velocity and enhanced adhesion, which is also observed experimentally [4 ]. In addition, artificially facilitating L-selectin clustering by antibody cross-linking has revealed its capacity to transduce intracellular signals, which alter, among other things, the binding activity of CD18 integrins [3 , 6 7 8 9 10 11 12 13 ].

Another interesting feature of L-selectin is that its surface expression is down-regulated rapidly by ectodomain shedding following the activation of neutrophils in vitro by various stimuli and by their extravasation into inflamed tissue [14 15 16 17 ]. L-selectin shedding is mediated primarily by a metalloprotease [18 , 19 ]. Indeed, a disintegrin and metalloprotease 17 (ADAM17) has been shown to mediate L-selectin cleavage directly by primary leukocytes [20 ], including mature macrophages and neutrophils [17 ]. Studies involving radiation chimeric mice with leukocytes deficient in functional ADAM17 and gene-targeted mice, which express noncleavable L-selectin, indicate that ectodomain shedding is important for regulating the receptor’s surface density and neutrophil infiltration into sites of inflammation [17 , 21 ].

Currently, there is nothing known about the dynamics of membrane redistribution of ADAM17 in relationship to L-selectin during neutrophil adhesion. To begin addressing this, we examined the diffusion of L-selectin and ADAM17 on activated neutrophils in real-time during their adhesion to purified E-selectin and IL-1β-activated endothelium under shear. We show that chemoattractant stimulation of neutrophils bound to an E-selectin substrate, as well as neutrophils attached to activated HUVEC, results in the directed clustering of L-selectin and ADAM17 to the trailing edge of cells. Of interest is ADAM17 and L-selectin redistribute at different rates, and the density shift by L-selectin occurs more rapidly. Our data provide the first demonstration that ADAM17 can redistribute and co-cluster rapidly with L-selectin by activated neutrophils attached to E-selectin. These data thus provide further mechanistic insight into the regulation of L-selectin during neutrophil adhesion.


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MATERIALS AND METHODS
 
Antibodies and other reagents
The anti-L-selectin mAb DREG-200 has been described previously [22 , 23 ]. Humanized DREG-200 (huDREG-200) was generously provided by PDL BioPharma (Fremont, CA, USA). The L-selectin mAb FMC46 conjugated to FITC was purchased from Dako North America (Carpenteria, CA, USA). FITC and PE-conjugated anti-L-selectin (LAM1-116) as well as biotinylated anti-CD45 and anti-CD55 were purchased from Ancell (Bayport, MN, USA). The anti-ADAM17 mAb M220 has been described previously [24 , 25 ] and was conjugated with Alexa 546 (Molecular Probes, Eugene, OR, USA), as per the manufacturer’s instructions. FITC-conjugated anti-CD45 was purchased from BD Biosciences PharMingen (San Diego, CA, USA). Unconjugated and biotin-conjugated F(ab')2 goat anti-mouse IgG, FITC-conjugated F(ab')2 goat anti-human IgG, and Cy3-conjugated streptavidin were purchased from Jackson ImmunoResearch (West Grove, PA, USA). QDot 655 streptavidin was purchased from Quantum Dot Corp. (Hayward, CA, USA). A human E-selectin-human IgG Fc chimeric construct (E-selectin/Fc) was purchased from R&D Systems (Minneapolis, MN, USA). The cytokine IL-1β was purchased from (PeproTech, Rocky Hill, NJ, USA). fMLP was purchased from Sigma-Aldrich (St. Louis, MO, USA).

Leukocyte isolation
Peripheral blood was collected from healthy donors in sodium heparin in accordance with approved protocols by the Institutional Review Board, Human Subjects Committee at the University of Minnesota (St. Paul, MN, USA), and at the University of California, Davis (Davis, CA, USA). Neutrophils were isolated by Ficoll-hypaque centrifugation, and cell viabilities were assessed by exclusion of the vital dye trypan blue, as described previously [3 , 5 ]. All media and buffers used for neutrophil isolation and incubations were sterile and tested for endotoxin.

Confocal microscopy
To visualize the cell surface distribution pattern of L-selectin and ADAM17 on neutrophils activated in suspension, freshly isolated neutrophils were cultured for various lengths of time at 37°C, as detailed in the text, in RPMI plus 5 mM HEPES (RPMI-H), in the presence or absence of fMLP (10 nM). Afterwards, the cells were washed in cold RPMI-H, fixed with 1% paraformaldehyde, and then treated with 1% normal goat serum in PBS to block nonspecific antibody interactions. The neutrophils were then sequentially stained with M220 (anti-ADAM17), biotin-conjugated F(ab')2 goat anti-mouse IgG, QDot 655 streptavidin, 10% normal mouse serum, and FITC-conjugated LAM1-116 (anti-L-selectin).

Analysis of the colocalization of ADAM17, CD45, or CD55 with L-selectin before and after its induced clustering by antibody-mediated cross-linking was adapted from our previous studies [3 , 5 ]. Briefly, freshly isolated neutrophils were stained sequentially with huDREG-200 and FITC-conjugated F(ab')2 goat anti-human IgG. The treated neutrophils were then incubated at 37°C for 30 min to facilitate L-selectin clustering. Afterwards, the neutrophils were fixed in 1% paraformaldehyde, treated with 1% normal goat serum in PBS to block nonspecific antibody interactions, and stained with the anti-ADAM17 mAb M220, followed by biotin-conjugated F(ab')2 goat anti-mouse IgG and QDot 655 streptavidin, 10% normal mouse serum, and then biotinylated anti-CD55 or biotinylated anti-CD45, followed by Cy3-conjugated streptavidin.

After the facilitation of L-selectin clustering, all antibody-staining steps described above were performed at 4°C, and the neutrophils were washed with RPMI-H between steps. Nonspecific antibody labeling was determined using the appropriate isotype negative control antibodies. The appropriately labeled cells were then applied to poly-L-lysine-coated coverslips and mounted with Vectashield Hard-Set mounting medium (Vector Laboratories, Burlingame, CA, USA). Fluorescence analysis was performed on an Olympus Fluoview FV500 laser-scanning confocal microscope (Olympus, Center Valley, PA, USA) using a 60x oil immersion objective. Images were recorded and processed using Multi-Point Time Lapse software. Use of the confocal microscope was made available through a National Center for Research Resources Shared Instrumentation Grant (#1 S10 RR16851).

Flow cytometry
To assess the effects of antibody cross-linking of L-selectin on its shedding efficiency, freshly isolated neutrophils suspended in RPMI-H were treated on ice with the PE-conjugated anti-L-selectin mAb LAM1-116 and then with or without F(ab')2 goat anti-mouse IgG. Cells were washed with RPMI-H between steps. Nonspecific antibody labeling was determined using an appropriate isotype negative control antibody. The treated neutrophils were then incubated at 37°C for 30 min in the presence or absence of fMLP (10 nM). Afterwards, the cells were washed in cold RPMI-H and fixed with 1% paraformaldehyde. The antibody-labeled cells were analyzed by flow cytometry (10,000 cells/sample) on a FACSCanto instrument (Becton Dickinson Immunocytometry Systems, San Jose, CA, USA).

Hydrodynamic shear flow assay
Prior to their use in the shear flow assays, neutrophils were kept at 4°C in a HEPES buffer [110 mM NaCl, 10 mM KCl, 10 mM glucose, 1 mM MgCl2, and 30 mM HEPES (pH 7.4)]. Immediately prior to the assay, CaCl2 was added to the buffer at a 1.5-mM final concentration. By this method, neutrophils were found to remain viable and unactivated for 4 h after their separation.

Microfluidic flow chambers with a minimum feature size of 5 µ were cast from Sylgard 184 prepolymer (Dow Corning, Midland, MI, USA) over a patterned silicon wafer and bonded to precleaned coverslips, which were coated with E-selectin/Fc in PBS containing 20 mM bicarbonate (1 µg/mL) for 90 min at room temperature. To block nonspecific binding sites, 2% human serum albumin (HSA) in PBS containing 0.05% Tween-20 was injected into the flow channel and incubated for an additional 90 min at room temperature. HUVEC were obtained from Cascade Biologics (Portland, OR, USA) and grown in Media 200 containing low-serum growth supplement (Cascade Biologics). At passages 4–5, HUVEC were layered onto the glass coverslips coated with cross-linked gelatin, grown to confluence, and then stimulated with 5 ng/mL IL-1β for 4 h, as described previously [26 ]. Finally, each flow channel was washed three times with PBS and connected to polyethylene tubing in preparation for shear flow experiments. To impose a specific shear stress, fluid was withdrawn from a reservoir through the flow chamber via a syringe pump (Harvard Apparatus, Holliston, MA, USA).

To label live neutrophils, the cells were incubated for 10 min at 4°C with 10 µg/mL of the L-selectin mAb FMC46-FITC (nonfunction blocking) or anti-CD45-FITC and the ADAM17 mAb M220 conjugated to Alexa 546 in HEPES buffer containing 1% HSA to block nonspecific antibody interactions. Following labeling, neutrophils were spun down and resuspended to 1 x 106/mL in HEPES buffer and then introduced into the microfluidic flow chamber. The average shear stress at the flow channel floor was set at 1 dyne/cm2 by adjusting the flow rate according to the Couette approximation (T=6 Qµ/wh2) [27 ]. To activate neutrophils rolling on E-selectin/Fc, the inlet reservoir fluid was replaced with 10 nM fMLP at a defined time-point. For all experiments, one fluorescent image was captured every 5 s by a Cascade 512B camera (Roper Scientific, Duluth, GA, USA) after sequential exposure using a filter wheel/shutter system on an inverted Nikon 1200 microscope (Nikon, Melville, NY, USA). A 50-frame sequence of three-color (Mean Red, Mean Green, and Mean Blue) images was reconstructed from each set of 488 nm, 546 nm, and brightfield channel data and saved for analysis. Image sequences were analyzed for the position of fluorescent clusters. Based on a histogram of all pixel intensities within an image region bounded by a neutrophil, clusters of ADAM17, L-selectin, or CD45 staining were defined as regions with pixel intensity, 2 SD greater than the mean neutrophil intensity. Using custom macros written in Image Pro 5.1 (Mediacybernetics, Silver Spring, MD, USA), these regions of dense protein were analyzed for percent area overlap in the red and green channels and cluster position.

Statistical analyses
Data analysis was performed using GraphPad Prism Version 4.0 software (GraphPad Software, San Diego, CA, USA). All data are reported as mean ± SD. Gaissian-distributed mean values were analyzed by Student’s t-test. Group comparisons were deemed significant for P values below 0.05.


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RESULTS
 
Distribution pattern of L-selectin and ADAM17 on the surface of activated neutrophils in suspension
L-selectin is distributed primarily on the tips of microvillous surface projections of resting leukocytes in suspension [28 ]. Upon cell activation, the distribution pattern of L-selectin on suspended cells remains essentially unchanged, but the number of surface molecules decreases rapidly as a result of their shedding [29 ]. Using confocal fluorescence microscopy, we examined the surface-staining pattern of anti-L-selectin and anti-ADAM17 mAb simultaneously by neutrophils activated in suspension with fMLP. L-selectin on unstimulated neutrophils demonstrated punctate but uniform staining (Fig. 1 ), as reported previously [3 , 5 ]. Upon cell activation, essentially the same distribution pattern for L-selectin was noted, but its staining level decreased to that of background within a few minutes (Fig. 1) . This did not occur in the absence of fMLP within the same time-frame (data not shown). ADAM17 also exhibited a punctate but uniform staining pattern on unstimulated and activated neutrophils, but unlike L-selectin, its staining level remained essentially unchanged following neutrophil activation (Fig. 1) . These findings indicate that ADAM17 and its substrate L-selectin do not undergo a striking topographical redistribution on the surface of neutrophils when activated in suspension.


Figure 1
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  		Figure 1. Cell surface distribution pattern of ADAM17 and L-selectin on neutrophils activated under static conditions. Freshly isolated human neutrophils under static conditions were treated with 10 nM fMLP for the indicated time-points. After which, the cells were dual-stained for L-selectin and ADAM17, as described in Materials and Methods. Confocal micrographs are representative of three independent experiments.

Spatial and temporal redistribution of L-selectin and ADAM17 in activated neutrophils during tethering on an E-selectin substrate under shear flow
Neutrophil attachment to E-selectin induces a rapid clustering of L-selectin and its eventual migration to the trailing edge of polarizing cells [3 4 5 ]. Using two-color, live cell immunofluorescence, we examined the cell surface distribution of L-selectin and ADAM17 simultaneously by fMLP-activated neutrophils adhered to E-selectin under shear flow. As described previously [3 ], L-selectin underwent a rapid redistribution to the trailing edge of E-selectin-bound neutrophils during their arrest and shape polarization (Fig. 2A ). The membrane distribution of ADAM17 changed as well, and its staining pattern shifted toward the trailing edge of E-selectin-attached neutrophils and began to occupy the L-selectin cluster (Fig. 2A and 2B) . In previous studies, we have found that a control surface receptor CD45 did not redistribute in the plasma membrane of neutrophils as efficiently as L-selectin upon E-selectin binding [3 , 5 ]. In the current study, we observed that CD45 underwent significantly less co-clustering with ADAM17 than did L-selectin during the same time-frame of E-selectin tethering (Fig. 2B) . Of particular interest was that the rates of L-selectin and ADAM17 redistribution to the uropod of polarizing neutrophils were significantly different, and the retrograde motion of L-selectin was more rapid (Fig. 2C) .


Figure 2
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  		Figure 2. ADAM17 and L-selectin undergo redistribution following the activation of neutrophils rolling on E-selectin. Freshly isolated human neutrophils were immunofluorescently labeled with nonfunction blocking, anti-L-selectin-FITC or anti-CD45-FITC, and anti-ADAM17-Alexa 546 mAb, as described in Materials and Methods. The labeled cells were perfused into a flow chamber and sheared over adsorbed E-selectin. After a constant shear stress for ≥1 min, fMLP (10 nM) was added to the inlet reservoir fluid, and the cells were monitored by immunofluorescence microscopy for the indicated times. (A) Image sequences indicate the position of fluorescent staining by L-selectin (green) and ADAM17 (red) on an individual adhered neutrophil, as indicated. One fluorescent image was captured every 5 s. (B) Cell micrographs were analyzed for the percentage of L-selectin or CD45 pixels overlapped by ADAM17 pixels (i.e., yellow pixels were divided by green and yellow pixels and then multiplied by 100). *, P = 0.005, versus (CD45 vs. ADAM17). (C) Cell micrographs were analyzed for L-selectin and ADAM17 staining concentrations in the trailing edge of activated neutrophils during their arrest, spread, and polarization. Micrograph pixel intensity for each labeled mAb was defined as 2.5 SD above average cellular fluorescence. *, P < 0.001; **, P = 0.008, versus ADAM17. (B and C) Data are given as mean ± SE for ≥15 cells per time-point for five independent experiments.

Neutrophils attached to stimulated endothelial monolayers undergo clustering of L-selectin as well [3 ]. IL-1β-stimulated HUVEC express high levels of E-selectin and chemokines [26 , 30 ], and we measured the redistribution and colocalization of L-selectin and ADAM17 on adhered neutrophils under shear flow. We observed that during the process of firm attachment and transmigration beneath the endothelial monolayer, ADAM17 and L-selectin clustered at the trailing edge of neutrophils, and the relocation of L-selectin occurred at a faster rate than ADAM17 (Fig. 3A and 3B ). Altogether, these data demonstrate that in contrast to neutrophils activated in suspension, L-selectin and ADAM17 undergo a coordinated lateral movement and focal coalescence in activated neutrophils rolling on E-selectin under shear flow, and the redistribution of L-selectin was more rapid than ADAM17.


Figure 3
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  		Figure 3. ADAM17 and L-selectin colocalize upon neutrophil transmigration through activated HUVEC. Freshly isolated human neutrophils were labeled immunofluorescently with nonfunction-blocking anti-L-selectin-FITC or anti-CD45-FITC and anti-ADAM17-Alexa 546 mAb, perfused into a flow chamber, and sheared over HUVEC monolayers, as described in Materials and Methods. (A) Image sequences indicate the position of fluorescent staining of L-selectin (green) and ADAM17 (red), as indicated on an individual transmigrating neutrophil, starting from the moment a subendothelial pseudopod is visible by brightfield microscopy. Upper panels represent the phase-contrast image of an attached neutrophil with overlaid fluorescence, and the lower panels display fluorescence overlap occurring in the L-selectin cluster in a pixilated manner. Values indicated in the lower panels represent the percentage of L-selectin pixels overlapped with ADAM17 pixels (i.e., yellow pixels were divided by green and yellow pixels and then multiplied by 100). (B) Cell micrographs were analyzed for the percentage of L-selectin or CD45 pixels overlapped by ADAM17 pixels. Micrograph pixel intensity for each labeled mAb was defined as 2.5 SD above average cellular fluorescence. Data are given as mean ± SE for ≥15 cells per time-point for five independent experiments. *, P = 0.008, versus (CD45 vs. ADAM17).

Effects of facilitated L-selectin clustering on the cell surface distribution of ADAM17 in neutrophils
We next examined whether the facilitated clustering of L-selectin on the surface of resting neutrophils resulted in a redistribution of ADAM17. Antibody cross-linking has been used extensively for specific and precise regulation of the extent of L-selectin clustering [5 , 6 , 12 , 31 32 33 34 35 36 ]. Using this approach, we examined whether ADAM17 co-clustered with antibody cross-linked L-selectin on resting neutrophils using confocal fluorescence microscopy. As we have reported previously [5 ], antibody cross-linking resulted in L-selectin patching on the neutrophil surface, which in turn, promoted appreciable clustering of CD55 (Fig. 4A h ), a GPI-anchored protein resident in leukocyte lipid rafts [37 38 39 40 ], but not CD45 (data not shown; ref. [5 ]). Moreover, the vast majority of the CD55 clusters colocalized with L-selectin (Fig. 4A i) . This degree of clustering or increased colocalization was not observed for ADAM17 upon antibody cross-linking of L-selectin (Fig. 4Ae and 4Af) . It is interesting that antibody cross-linking of L-selectin greatly diminished its down-regulation in surface expression upon neutrophil activation with fMLP compared with noncross-linked L-selectin on stimulated neutrophils (Fig. 4B) , further indicating that L-selectin clustering in this manner sequestered it from ADAM17. The above findings thus suggest that ADAM17 and L-selectin are not associated in a physical manner in unactivated neutrophils, nor do they occupy a common membrane microdomain.


Figure 4
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  		Figure 4. Antibody-mediated cross-linking of L-selectin in resting neutrophils does not facilitate the clustering of ADAM17. (A) Freshly isolated neutrophils were treated with an L-selectin mAb and FITC-conjugated secondary antibody and incubated on ice (a–c) or at 37°C for 30 min (d–i) to coalesce L-selectin (L-selectin-XL). After which, the neutrophils were stained for CD55 or ADAM17, as indicated. Neutrophils incubated on ice demonstrated uniform, punctate staining of L-selectin (a). Neutrophils incubated at 37°C demonstrated increased patching of L-selectin (d and g). A merged image of L-selectin and CD55 (i) shows several yellow areas (some are indicated by arrowheads). Values indicated in the merged panels represent the percentage of ADAM17 or CD55 pixels overlapped with L-selectin pixels (i.e., yellow pixels were divided by red and yellow pixels and then multiplied by 100). Confocal micrographs are representative of 25 cells from three independent experiments. (B) Freshly isolated neutrophils were treated with a fluorochrome-conjugated L-selectin mAb, plus or minus F(ab')2 goat anti-mouse IgG, and then incubated for 15 min at 37°C [only the former treatment promoted L-selectin clustering (data not shown)], with or without 10 mM fMLP, as indicated. Neutrophils treated with a fluorochrome-conjugated L-selectin mAb, plus or minus F(ab')2 goat anti-mouse IgG at 37°C in the absence of fMLP, demonstrated equivalent mean fluorescent cell-staining levels (data not shown). Cell staining was examined by flow cytometry, and 10,000 cells were examined per sample. The dashed line indicates cell staining by a fluorochrome-conjugated, isotype-matched, negative control mAb. Data are representative of three independent experiments.


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DISCUSSION
 
L-selectin undergoes rapid clustering on the surface of neutrophils upon its engagement and rolling on a cell substrate expressing E-selectin [3 , 4 ]. L-selectin is also down-regulated promptly in expression by the metalloprotease ADAM17 following overt activation of neutrophils by various stimuli and during their extravasation into inflamed tissues [14 15 16 17 ]. At this time, the distribution pattern of ADAM17 in relationship to L-selectin, while the adhesion molecule undergoes clustering during neutrophil tethering on E-selectin, has not been determined. We simultaneously imaged the distribution of L-selectin and ADAM17 on live human neutrophils upon their rolling and activation on purified E-selectin and inflamed vascular endothelial cells under shear flow. Of interest is that ADAM17 also underwent membrane redistribution and progressed to polarized clusters at the trailing edge of neutrophils, although at an apparently slower rate than L-selectin. Such striking focal colocalization of L-selectin and ADAM17 was not apparent on neutrophils activated in suspension. These findings reveal the involvement of additional mechanisms in the regulation of L-selectin activity and its shedding by adherent leukocytes under shear flow conditions.

The clustering of L-selectin increases its valency [4 , 41 , 42 ] and transduces cellular signals from the outside in, resulting in various post-L-selectin adhesion events, including oxidative burst, degranulation, cytokine expression, actin polymerization, and CD18 integrin activation [3 , 6 , 8 , 12 , 31 32 33 34 35 36 , 43 ]. Hence, L-selectin clustering appears to promote various leukocyte effector activities prior to its down-regulation by ectodomain shedding. L-selectin clustering may also be important in enhancing its proteolytic turnover by increasing the proximity of L-selectin molecules to facilitate more efficient shedding. A limitation of the fluorescent imaging approaches used in our study is a spatial resolution cutoff of approximately 0.02 µm to assess molecular scale interactions between L-selectin and ADAM17. However, approaches such as electron microscopy, which can assess L-selectin and ADAM17 intermixing at the ultrastructural level, or fluorescence resonance energy transfer, which detects signal emissions dependent on intermolecular proximity, present various technical challenges when real-time imaging live cells in our microfluidic flow chamber. Although not measured directly, L-selectin shedding by adherent neutrophils may begin prior to its redistribution. In consideration of this, it is tempting to speculate that the observed redistribution of L-selectin molecules ahead of ADAM17 in tethered neutrophils may delay their shedding upon clustering in the uropod by altering the stoichiometry of the proteolytic reaction or temporarily sequestering L-selectin from its sheddase. Consistent with the latter assumption, we observed that antibody-mediated L-selectin clustering greatly reduced the level of L-selectin down-regulation upon neutrophil activation with fMLP when compared with neutrophils that were subjected to activation only.

It has been reported that L-selectin clustering in neutrophils upon their binding to E-selectin involves an active transport process [3 ]. Indeed, the lateral mobility of L-selectin appears to be regulated by dynamic associations with membrane domains and the actin cytoskeleton [5 , 44 , 45 ]. The topographical redistribution of ADAM17 during this process may also involve an active transport process. This assumption is based on several findings. ADAM17 and L-selectin appear not to associate in a physical manner or partition in a common membrane domain, either of which might facilitate passive diffusion by ADAM17 upon the coalescence of L-selectin, as appears to occur for CD55 [3 ]. L-selectin and ADAM17 progressed to the trailing edge of activated neutrophils during adhesion and transmigration at different rates under shear flow conditions. Lastly, under static conditions, ADAM17, but not L-selectin, demonstrated little redistribution to the uropod of neutrophils undergoing transmigration through activated HUVEC (data not shown). These findings together indicate that L-selectin and ADAM17 are not constitutively associated and exhibit distinct membrane transport properties. Canault et al. [46 ] reported that four-and-a-half LIM domain 2 protein, which is involved in various protein-binding interactions, associates with the cytoplasmic region of ADAM17 and the actin cytoskeleton. This and other intermolecular interactions may facilitate cytoskeletal linkages and lateral movement by ADAM17. It will be important to gain a better understanding of the mechanisms that direct the lateral movement of surface ADAM17, as this may provide for additional means to manipulate the activity of ADAM17 during leukocyte extravasation into sites of inflammation.

In conclusion, when neutrophils bind inflamed vascular endothelial cells, they become polarized, and L-selectin congregates at their trailing edge. We show that this directed clustering occurs for ADAM17 as well. When adhered to E-selectin, activated neutrophils redistribute first L-selectin and then ADAM17 to the uropod in a time course consistent with the onset of L-selectin shedding. Such redistribution may facilitate transendothelial migration in several ways. First, the redistribution of L-selectin may decrease adhesion events at the neutrophil’s leading edge. Second, L-selectin clustering by attached neutrophils provides potent inside-out signaling, resulting in a conformational shift in the CD18 integrins, which leads to neutrophil deceleration and arrest [3 , 9 , 12 ]. Finally, ADAM17 redistribution to the uropod may provide a means for extinguishing L-selectin adhesion events and outside-in signaling, facilitating a smoother transition from surface adhesion to interstitial migration. In support of this theory, Venturi et al. [21 ] reported that neutrophils in gene-targeted mice expressing noncleavable L-selectin were impaired in their transendothelial migration across postcapillary venules during keratinocyte-derived cytokine-induced inflammation. We thus conclude that a proteolytic step supplied by ADAM17 redistribution in relation to L-selectin aids in the precision of the process of rolling, activation, arrest, and transmigration by neutrophils.


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ACKNOWLEDGEMENTS
 
This study was supported by grants HL61613 (B. W.) and AI47294 (S. I. S.) from the National Institutes of Health. We thank Drs. Roy Black and Jacques Peschon for providing the anti-human ADAM17 mAb M220 and Drs. Zhenya Ni and Josh Mattila for their technical assistance with Photoshop.


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FOOTNOTES
 
1 These authors contributed equally to this work. Back

Received May 16, 2007; revised September 17, 2007; accepted September 17, 2007.


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REFERENCES
 
    1
  1. Rainer, T. H. (2002) L-selectin in health and disease Resuscitation 52,127-141[CrossRef][Medline]
    2. 2
  2. Zollner, O., Lenter, M. C., Blanks, J. E., Borges, E., Steegmaier, M., Zerwes, H. G., Vestweber, D. (1997) L-selectin from human, but not from mouse neutrophils binds directly to E-selectin J. Cell Biol. 136,707-716[Abstract/Free Full Text]
    3. 3
  3. Green, C. E., Pearson, D. N., Camphausen, R. T., Staunton, D. E., Simon, S. I. (2004) Shear-dependent capping of L-selectin and P-selectin glycoprotein ligand 1 by E-selectin signals activation of high-avidity β2-integrin on neutrophils J. Immunol. 172,7780-7790[Abstract/Free Full Text]
    4. 4
  4. King, M. R., Sumagin, R., Green, C. E., Simon, S. I. (2005) Rolling dynamics of a neutrophil with redistributed L-selectin Math. Biosci. 194,71-79[CrossRef][Medline]
    5. 5
  5. Mattila, P. E., Green, C. E., Schaff, U., Simon, S. I., Walcheck, B. (2005) Cytoskeletal interactions regulate inducible L-selectin clustering Am. J. Physiol. Cell Physiol. 289,C323-C332[Abstract/Free Full Text]
    6. 6
  6. Crockett-Torabi, E., Sulenbarger, B., Smith, C. W., Fantone, J. C. (1995) Activation of human neutrophils through L-selectin and Mac-1 molecules J. Immunol. 154,2291-2302[Abstract]
    7. 7
  7. Simon, S. I., Burns, A. R., Taylor, A. D., Gopalan, P. K., Lynam, E. B., Sklar, L. A., Smith, C. W. (1995) L-selectin (CD62L) cross-linking signals neutrophil adhesive functions via the Mac-1 (CD11b/CD18) β 2-integrin J. Immunol. 155,1502-1514[Abstract]
    8. 8
  8. Steeber, D. A., Engel, P., Miller, A. S., Sheetz, M. P., Tedder, T. F. (1997) Ligation of L-selectin through conserved regions within the lectin domain activates signal transduction pathways and integrin function in human, mouse, and rat leukocytes J. Immunol. 159,952-963[Abstract]
    9. 9
  9. Tsang, Y. T., Neelamegham, S., Hu, Y., Berg, E. L., Burns, A. R., Smith, C. W., Simon, S. I. (1997) Synergy between L-selectin signaling and chemotactic activation during neutrophil adhesion and transmigration J. Immunol. 159,4566-4577[Abstract]
    10. 10
  10. Simon, S. I., Cherapanov, V., Nadra, I., Waddell, T. K., Seo, S. M., Wang, Q., Doerschuk, C. M., Downey, G. P. (1999) Signaling functions of L-selectin in neutrophils: alterations in the cytoskeleton and colocalization with CD18 J. Immunol. 163,2891-2901[Abstract/Free Full Text]
    11. 11
  11. Simon, S. I., Hu, Y., Vestweber, D., Smith, C. W. (2000) Neutrophil tethering on E-selectin activates β 2 integrin binding to ICAM-1 through a mitogen-activated protein kinase signal transduction pathway J. Immunol. 164,4348-4358[Abstract/Free Full Text]
    12. 12
  12. Green, C. E., Pearson, D. N., Christensen, N. B., Simon, S. I. (2003) Topographic requirements and dynamics of signaling via L-selectin on neutrophils Am. J. Physiol. Cell Physiol. 284,C705-C717[Abstract/Free Full Text]
    13. 13
  13. McDonough, D. B., McIntosh, F. A., Spanos, C., Neelamegham, S., Goldsmith, H. L., Simon, S. I. (2004) Cooperativity between selectins and β2-integrins define neutrophil capture and stable adhesion in shear flow Ann. Biomed. Eng. 32,1179-1192[CrossRef][Medline]
    14. 14
  14. Kishimoto, T. K., Jutila, M. A., Berg, E. L., Butcher, E. C. (1989) Neutrophil Mac-1 and MEL-14 adhesion proteins inversely regulated by chemotactic factors Science 245,1238-1241[Abstract/Free Full Text]
    15. 15
  15. Jutila, M. A., Rott, L., Berg, E. L., Butcher, E. C. (1989) Function and regulation of the neutrophil MEL-14 antigen in vivo: comparison with LFA-1 and Mac-1 J. Immunol. 143,3318-3324[Abstract]
    16. 16
  16. Kahn, J., Ingraham, R. H., Shirley, F., Migaki, G. I., Kishimoto, T. K. (1994) Membrane proximal cleavage of L-selectin: identification of the cleavage site and a 6-kD transmembrane peptide fragment of L- selectin J. Cell Biol. 125,461-470[Abstract/Free Full Text]
    17. 17
  17. Li, Y., Brazzell, J., Herrera, A., Walcheck, B. (2006) ADAM17 deficiency by mature neutrophils has differential effects on L-selectin shedding Blood 108,2275-2279[Abstract/Free Full Text]
    18. 18
  18. Walcheck, B., Kahn, J., Fisher, J. M., Wang, B. B., Fisk, R. S., Payan, D. G., Feehan, C., βgeri, R., Darlak, K., Spatola, A. F., Kishimoto, T. K. (1996) Neutrophil rolling altered by inhibition of L-selectin shedding in vitro Nature 380,720-723[CrossRef][Medline]
    19. 19
  19. Walcheck, B., Alexander, S. R., St Hill, C. A., Matala, E. (2003) ADAM-17-independent shedding of L-selectin J. Leukoc. Biol. 74,389-394[Abstract/Free Full Text]
    20. 20
  20. Peschon, J. J., Slack, J. L., Reddy, P., Stocking, K. L., Sunnarborg, S. W., Lee, D. C., Russell, W. E., Castner, B. J., Johnson, R. S., Fitzner, J. N., Boyce, R. W., Nelson, N., Kozlosky, C. J., Wolfson, M. F., Rauch, C. T., Cerretti, D. P., Paxton, R. J., March, C. J., Black, R. A. (1998) An essential role for ectodomain shedding in mammalian development Science 282,1281-1284[Abstract/Free Full Text]
    21. 21
  21. Venturi, G. M., Tu, L., Kadono, T., Khan, A. I., Fujimoto, Y., Oshel, P., Bock, C. B., Miller, A. S., Albrecht, R. M., Kubes, P., Steeber, D. A., Tedder, T. F. (2003) Leukocyte migration is regulated by L-selectin endoproteolytic release Immunity 19,713-724[CrossRef][Medline]
    22. 22
  22. Kishimoto, T. K., Jutila, M. A., Butcher, E. C. (1990) Identification of a human peripheral lymph node homing receptor: a rapidly down-regulated adhesion molecule Proc. Natl. Acad. Sci. USA 87,2244-2248[Abstract/Free Full Text]
    23. 23
  23. Matala, E., Alexander, S. R., Kishimoto, T. K., Walcheck, B. (2001) The cytoplasmic domain of L-selectin participates in regulating L-selectin endoproteolysis J. Immunol. 167,1617-1623[Abstract/Free Full Text]
    24. 24
  24. Doedens, J. R., Black, R. A. (2000) Stimulation-induced down-regulation of tumor necrosis factor-{alpha} converting enzyme J. Biol. Chem. 275,14598-14607[Abstract/Free Full Text]
    25. 25
  25. Walcheck, B., Herrera, A. H., Hill, C. S., Mattila, P. E., Whitney, A. R., Deleo, F. R. (2006) ADAM17 activity during human neutrophil activation and apoptosis Eur. J. Immunol. 36,968-976[CrossRef][Medline]
    26. 26
  26. Gopalan, P. K., Smith, C. W., Lu, H., Berg, E. L., McIntire, L. V., Simon, S. I. (1997) Neutrophil CD18-dependent arrest on intercellular adhesion molecule 1 (ICAM-1) in shear flow can be activated through L-selectin J. Immunol. 158,367-375[Abstract]
    27. 27
  27. Green, C. E., Schaff, U. Y., Sarantos, M. R., Lum, A. F., Staunton, D. E., Simon, S. I. (2006) Dynamic shifts in LFA-1 affinity regulate neutrophil rolling, arrest, and transmigration on inflamed endothelium Blood 107,2101-2111[Abstract/Free Full Text]
    28. 28
  28. Erlandsen, S. L., Hasslen, S. R., Nelson, R. D. (1993) Detection and spatial distribution of the β2 integrin (Mac-1) and L-selectin (LECAM-1) adherence receptors on human neutrophils by high-resolution field emission SEM J. Histochem. Cytochem. 41,327-333[Abstract]
    29. 29
  29. Fors, B. P., Goodarzi, K., von Andrian, U. H. (2001) L-selectin shedding is independent of its subsurface structures and topographic distribution J. Immunol. 167,3642-3651[Abstract/Free Full Text]
    30. 30
  30. Yoshida, M., Westlin, W. F., Wang, N., Ingber, D. E., Rosenzweig, A., Resnick, N., Gimbrone, M. A., Jr (1996) Leukocyte adhesion to vascular endothelium induces E-selectin linkage to the actin cytoskeleton J. Cell Biol. 133,445-455[Abstract/Free Full Text]
    31. 31
  31. Laudanna, C., Constantin, G., Baron, P., Scarpini, E., Scarlato, G., Cabrini, G., Dechecchi, C., Rossi, F., Cassatella, M. A., Berton, G. (1994) Sulfatides trigger increase of cytosolic free calcium and enhanced expression of tumor necrosis factor-{alpha} and interleukin-8 mRNA in human neutrophils. Evidence for a role of L- selectin as a signaling molecule J. Biol. Chem. 269,4021-4026[Abstract/Free Full Text]
    32. 32
  32. Waddell, T. K., Fialkow, L., Chan, C. K., Kishimoto, T. K., Downey, G. P. (1994) Potentiation of the oxidative burst of human neutrophils. A signaling role for L-selectin J. Biol. Chem. 269,18485-18491[Abstract/Free Full Text]
    33. 33
  33. Simon, S. I., Burns, A. R., Taylor, A. D., Gopalan, P. K., Lynam, E. B., Sklar, L. A., Smith, C. W. (1995) L-selectin (CD62L) cross-linking signals neutrophil adhesive functions via the Mac-1 (CD11b/CD18) β2-integrin J. Immunol. 155,1502-1514[Abstract]
    34. 34
  34. Waddell, T. K., Fialkow, L., Chan, C. K., Kishimoto, T. K., Downey, G. P. (1995) Signaling functions of L-selectin: enhancement of tyrosine phosphorylation and activation of MAP kinase J. Biol. Chem. 270,15403-15411[Abstract/Free Full Text]
    35. 35
  35. Taylor, A. D., Neelamegham, S., Hellums, J. D., Smith, C. W., Simon, S. I. (1996) Molecular dynamics of the transition from L-selectin- to β 2-integrin-dependent neutrophil adhesion under defined hydrodynamic shear Biophys. J. 71,3488-3500[Medline]
    36. 36
  36. Smolen, J. E., Petersen, T. K., Koch, C., O’Keefe, S. J., Hanlon, W. A., Seo, S., Pearson, D., Fossett, M. C., Simon, S. I. (2000) L-selectin signaling of neutrophil adhesion and degranulation involves p38 mitogen-activated protein kinase J. Biol. Chem. 275,15876-15884[Abstract/Free Full Text]
    37. 37
  37. Parolini, I., Sargiacomo, M., Lisanti, M. P., Peschle, C. (1996) Signal transduction and glycophosphatidylinositol-linked proteins (lyn, lck, CD4, CD45, G proteins, and CD55) selectively localize in Triton-insoluble plasma membrane domains of human leukemic cell lines and normal granulocytes Blood 87,3783-3794[Abstract/Free Full Text]
    38. 38
  38. Kwiatkowska, K., Sobota, A. (2001) The clustered Fc{gamma} receptor II is recruited to Lyn-containing membrane domains and undergoes phosphorylation in a cholesterol-dependent manner Eur. J. Immunol. 31,989-998[CrossRef][Medline]
    39. 39
  39. Leitinger, B., Hogg, N. (2002) The involvement of lipid rafts in the regulation of integrin function J. Cell Sci. 115,963-972[Abstract/Free Full Text]
    40. 40
  40. Frasch, S. C., Henson, P. M., Nagaosa, K., Fessler, M. B., Borregaard, N., Bratton, D. L. (2004) Phospholipid flip-flop and phospholipid scramblase 1 (PLSCR1) co-localize to uropod rafts in formylated Met-Leu-Phe-stimulated neutrophils J. Biol. Chem. 279,17625-17633[Abstract/Free Full Text]
    41. 41
  41. Li, X., Steeber, D. A., Tang, M. L., Farrar, M. A., Perlmutter, R. M., Tedder, T. F. (1998) Regulation of L-selectin-mediated rolling through receptor dimerization J. Exp. Med. 188,1385-1390[Abstract/Free Full Text]
    42. 42
  42. Dwir, O., Steeber, D. A., Schwarz, U. S., Camphausen, R. T., Kansas, G. S., Tedder, T. F., Alon, R. (2002) L-selectin dimerization enhances tether formation to properly spaced ligand J. Biol. Chem. 277,21130-21139[Abstract/Free Full Text]
    43. 43
  43. Junge, S., Brenner, B., Lepple-Wienhues, A., Nilius, B., Lang, F., Linderkamp, O., Gulbins, E. (1999) Intracellular mechanisms of L-selectin induced capping Cell. Signal. 11,301-308[CrossRef][Medline]
    44. 44
  44. Ivetic, A., Deka, J., Ridley, A., Ager, A. (2002) The cytoplasmic tail of L-selectin interacts with members of the ezrin- radixin-moesin (ERM) family of proteins: cell activation-dependent binding of moesin but not ezrin J. Biol. Chem. 277,2321-2329[Abstract/Free Full Text]
    45. 45
  45. Ivetic, A., Florey, O., Deka, J., Haskard, D. O., Ager, A., Ridley, A. J. (2004) Mutagenesis of the ezrin-radixin-moesin binding domain of L-selectin tail affects shedding, microvillar positioning, and leukocyte tethering J. Biol. Chem. 279,33263-33272[Abstract/Free Full Text]
    46. 46
  46. Canault, M., Tellier, E., Bonardo, B., Mas, E., Aumailley, M., Juhan-Vague, I., Nalbone, G., Peiretti, F. (2006) FHL2 interacts with both ADAM-17 and the cytoskeleton and regulates ADAM-17 localization and activity J. Cell. Physiol. 208,363-372[CrossRef][Medline]



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Y. Wang, A. H. Herrera, Y. Li, K. K. Belani, and B. Walcheck
Regulation of Mature ADAM17 by Redox Agents for L-Selectin Shedding
J. Immunol., February 15, 2009; 182(4): 2449 - 2457.
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