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Blood, Vol. 95 No. 8 (April 15), 2000:
pp. 2471-2480
Oriented endocytic recycling of  5 1 in motile neutrophils
Lynda M. Pierini,
Moira A. Lawson,
Robert J. Eddy,
Bill Hendey, and
Frederick R. Maxfield
From the Department of Biochemistry, Weill Medical College of
Cornell University, New York, NY; Department of Dairy and Food Science,
The Royal Veterinary and Agricultural University, Frederiksberg,
Denmark; and Department of Pharmacology, Rush Medical College,
Rush-Presbyterian-St. Luke's-Medical Center, Chicago, IL.
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Abstract |
During cell migration, integrin attachments to the substratum
provide the means to generate the traction and force necessary to
achieve locomotion. Once the cell has moved over these attachments, however, it is equally important that integrins detach from the substratum. The fate of integrins after detachment may include release
from the cell, lateral diffusion across the cell surface, or
endocytosis and redelivery to the cell surface. Polymorphonuclear neutrophils (PMNs) become stuck on the extracellular matrix proteins fibronectin and vitronectin when their intracellular free calcium concentration ([Ca++]i) is buffered.
Taking advantage of this feature of PMN migration, we investigated the
fate of integrins to differentiate among various models of migration.
We demonstrate that  5 1, one of the fibronectin-binding integrins,
is responsible for immobilization of
[Ca++]i-buffered PMNs on fibronectin. We
find that 5 and 1 are in endocytic vesicles in PMNs and that 5
colocalizes with a marker for an endocytic recycling compartment. When
[Ca++]i is buffered, 5 and 1 become
concentrated in clusters in the rear of the adherent cells, suggesting
that [Ca++]i transients are required for
51 detachment from the substratum. Inhibition of 51
detachment by buffering [Ca++]i results
in the depletion of 5 from both endocytic vesicles and the recycling
compartment, providing compelling evidence that integrins are normally
recycled by way of endocytosis and intracellular trafficking during
cell migration. This model is further refined by our demonstration that
the endocytic recycling compartment reorients to retain its
localization just behind the leading lamella as PMNs migrate,
indicating that membrane recycling during neutrophil migration has directionality.
(Blood. 2000;95:2471-2480)
© 2000 by The American Society of Hematology.
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Introduction |
Neutrophils migrate from the blood stream, through the
vascular endothelium and connective tissue to sites of inflammation or
infection. This process occurs after stimulation by chemoattractants generated by bacteria (eg, N-formylated peptides) or the immune system
(eg, complement component C5a). After chemoattractant stimulation, neutrophils activate several intracellular signaling
pathways,1 including rapid and repeated changes in the
intracellular free calcium concentration
([Ca++]i).2,3 Previous studies
have shown that these transients are required for motility on
fibronectin and vitronectin,4,5 substrates encountered in
the connective tissue stroma. On vitronectin, this loss of motility has
been shown to be due to the clustering of  v3 integrin in the rear
of [Ca++]i-buffered cells.6 Both
the loss of motility and the clustering of v3 found in
[Ca++]i-buffered neutrophils on vitronectin
can be mimicked by the addition of inhibitors of the serine/threonine
phosphatase, calcineurin.7 Although it has been suggested
that serine/threonine phosphatase inhibitors may play a role in the
attachment events of some fibronectin-binding proteins,8
the motility of neutrophils on fibronectin is not affected by
calcineurin inhibitory peptides.7
Neutrophils contain a number of integrins, of which the best
characterized belong to the 2 (CD18) family.9,10
Integrins in this family are involved in the attachment of neutrophils
to the endothelium,11 as well as attachment to a number of
proteins in the connective tissue stroma.12-14 Neutrophils
have also been shown to contain 51 integrin15,16,42
that is known to bind to fibronectin with a high affinity.
During neutrophil migration on a flat surface, the cell sends out
numerous pseudopods, some of which adhere to the substrate. The body of
the cell then proceeds forward in the direction of the newly formed
attachment. To continue moving, the cell must make attachments to the
substrate that can be released as it moves forward.5,17
There are several mechanisms used by cells to affect this release
without actively regulating integrin/substrate interactions:1 by using interactions that are reversible
over the time required for a cell to move forward over an attachment site,18-20 by leaving behind pieces of adherent membrane as
the cell moves forward,21,22 or by digesting the
extracellular matrix proteins to which it is attached.23
While migrating neutrophils use these unregulated mechanisms for some
adhesive interactions, they actively regulate their interactions with
vitronectin and fibronectin, using transient increases in
[Ca++]i to disrupt tight integrin/substrate
interactions so that the cells can continue moving.5,24
Various fates are plausible for integrins that undergo regulated
release from the substrate. It has been proposed in the case of
fibroblasts that newly released integrins disperse on the cell surface
to be used again in adhesions toward the front of the cell.21,25 Another possibility is that newly released
integrins become endocytosed, then transported to the cell surface
where they can diffuse to sites of attachment. A related possibility is
that integrins are endocytosed at the cell rear and are transported in
a directed manner toward the cell front.26,27 This oriented recycling of integrins, whereby endocytosis occurs near the uropod and
exocytosis occurs near the leading lamellae, provides a possible mechanism by which a cell could maintain a gradient of adhesiveness along its axis.
In a previous study, we used a shearing procedure,28 which
removes the upper surface of the cell and leaves behind only the lower
adherent membrane. In this way, proteins directly involved in
cell-substrate interactions were studied. For polymorphonuclear neutrophils (PMNs) crawling on vitronectin, we found that v3 integrins were present primarily near the leading edge on the adherent
membrane of polarized cells.6 Confocal microscopy on whole
cells showed that v3 integrins were also in endocytic vesicles.
When these cells were loaded with the cytoplasmic calcium buffer quin2,
the v3 integrins were found in clusters on the adherent
membrane at the rear of the cell. These clusters were also found in
cells in which calcineurin was inhibited, suggesting that calcium was
acting through calcineurin to break up integrin clusters.
In this paper, we examined which integrin on PMNs is responsible for
[Ca++]i-sensitive adhesion on fibronectin. We
show that antibodies to 51 integrins, but not against v3
integrins, restore motility to
[Ca++]i-buffered cells on fibronectin. We
also show that 5 integrins become clustered at the rear of
[Ca++]i-buffered cells on fibronectin. We
directly demonstrate that 5 integrins are internalized in motile
PMNs and are found colocalized with a marker of an endocytic recycling
compartment (ERC), strongly suggesting that integrins are indeed
recycled via endocytosis during PMN migration. Finally, using a
fluorescent label of the ERC, we establish that the ERC is located
toward the front of polarized PMNs and reorients during migration to
maintain this localization.
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Materials and methods |
Materials
Fibronectin and vitronectin were purchased from GIBCO BRL
(Gaithersburg, MD). Monoclonal antibodies CLB-705(5), JB1a(1), JB55(51), CLB-701(6), and MAB1962 (2) were purchased from Chemicon (Temecula, CA). LM609 (v3), IB4 (2), and MAB44 (2) were gifts from D. Cheresh (Scripps Research Institute, La Jolla, CA),
S. D. Wright (Merck Research Laboratories, Rahway, NJ), and A. Huttenlocher (University of Illinois at Urbana-Champaign, Urbana, IL),
respectively. Fluorescent-labeled secondary antibodies were purchased
from Pierce Scientific (Rockford, IL). Quin2/AM was purchased from
Molecular Probes (Eugene, OR). The calcineurin inhibitory peptide,
CN412, was a gift from C. Klee (NIH, Bethesda, MD).
N-formyl-methyl-leucyl-phenylalanine (fMLP) was purchased from Sigma
(St Louis, MO).
Neutrophil isolation
Polymorphonuclear leukocytes (neutrophils) were isolated from whole
blood donated by healthy volunteers by a single-step separation over a
ficoll-hypaque solution (GIBCO BRL). Contaminating erythrocytes were
lysed by a 30-second hypotonic shock. Cells were then rinsed with
phosphate-buffered saline (PBS) and resuspended in incubation buffer
(150 mmol/L NaCl, 5 mmol/L KCl, 1 mmol/L MgCl2, 10 mmol/L glucose, 20 mmol/L HEPES pH 7.4).
Intracellular calcium buffering
For calcium-buffering experiments, neutrophils were incubated in the
presence of 50 µmol/L quin2/AM solution as described previously.5 Briefly, the solution was prepared by adding 3 µL of a 50 µmol/L quin2/AM stock solution in anhydrous DMSO to 4 µL of a 25% w/v solution of pluronic-F127 in water. This mixture was
then added to 60 µL of heat-inactivated fetal calf serum, followed by
2.9 mL of incubation medium with mixing. The solution was then added to
3 mL of incubation medium containing 5 × 106
cells/mL. The cells were incubated in this mixture with gentle mixing
for 40 minutes at room temperature. After incubation, the cells were
washed twice in PBS and resuspended in incubation medium. After this
treatment, Ca++ transients stop and the basal
[Ca++]i levels drop to approximately 100 nmol/L.2 We can mimic the effects of quin2/AM buffering by
other Ca++-chelating agents (eg, BAPTA/AM), as well as by
keeping the PMNs in Ca++-free medium in the presence of
EGTA.2,24 The effects of quin2/AM on motility can be
attributed to the Ca++-buffering properties of quin2 as
opposed to the acetoxymethylester because loading cells with high
concentrations of quene-1/AM, a pH-sensitive relative of quin2/AM, has
no effect on neutrophil migration.2
Calcineurin inhibition
A peptide inhibitor of calcineurin, CN412, was delivered to the
cytoplasm by an endocytosis/hypo-osmotic shock procedure.7 Cells were incubated with 100 µmol/L CN412 for 30 minutes at 37°C to allow endocytosis of the calcineurin inhibitor peptide. Cells were
then subjected to osmotic shock in water without added salts for 30 seconds to disrupt endocytic vesicles, thereby introducing the peptide
into the cytoplasm. Control cells were subjected to osmotic shock in
the absence of the peptide. The cells were then rinsed thoroughly with
PBS and resuspended in incubation buffer. After the osmotic shock, an
estimated 1% to 5% of the external concentration of the peptide is
present in the cytoplasm.7 Successful CN412 loading was
verified by confirming that migration on vitronectin was inhibited in
the same batch of cells (data not shown).
Motility assays
Neutrophils were maintained in incubation buffer at 15°C to
prevent clumping and loss of the cytoplasmic CN412 or Quin2.
Neutrophils (103-104 cells) were then incubated ± antibodies for 15 minutes at 37°C before starting the
experiment. The neutrophils were plated onto the glass coverslip area
of the experimental chamber,24 which had been previously
coated with fibronectin (0.1 mg/mL) for 1 hour. The neutrophils were
maintained at 37°C and allowed to attach for 5 minutes. The medium
was removed and replaced with incubation buffer with or without 5 µg/mL antibody. After 5 minutes, the chemoattractant fMLP (10 nmol/L)
was added. The experimental chamber was then placed on a microscope
stage maintained at 37°C. Five minutes after the application of
fMLP, cell motility was monitored in the continued presence of antibody
or peptide using a Leitz Diavert microscope (Wetzlar, Germany) equipped
with Nomarski differential interference contrast (DIC)
optics. A video camera (CCD-72; Dage-MTI Inc, Michigan
City, IN) and an optical memory disk recorder (Panasonic; Matsushita
Electronics Corp, Osaka, Japan) were used to record single frames every
10 seconds for a period of 200 seconds. Migrating cells were defined as
those in which both the leading edge and tail of the cell were observed
to move at least 7 µm from their initial position in 200 seconds.24 Separate dishes were used for each treatment,
and 3 sequential fields were recorded from each dish. In most
experiments, 2 or more dishes were used for each treatment condition.
The percentage motile cells (number of motile cells divided by the
number of cells observed) was determined for each treatment group in
each experiment. Experiments were repeated with fresh preparations of
neutrophils on several days.
Flow cytometry
To verify 51 integrin expression on PMNs, cells were incubated
in incubation buffer containing saturating amounts of primary monoclonal antibody in the presence of 10% normal goat serum ± 10 nmol/L fMLP for 1 hour on ice. Binding experiments were used to
determine the concentration of each antibody required for saturation of
all binding sites on the cells (not shown). The cells were rinsed with
PBS and incubated in 5 µg/mL FITC-goat antimouse IgG for 1 hour on
ice. The cells were then rinsed well with PBS. For all flow cytometry
measurements, PMNs were resuspended at 1 × 106
cells/mL, and fluorescence was measured using a FACScan (Becton Dickinson, San Jose, CA) flow cytometer. Cell analysis was gated on forward and side scatter. The fluorescence intensity of PMNs incubated with the control antibody (MOPC21, dotted lines) was set to
an arbitrary number and all other samples were measured relative to
this value. In this way, contributions from nonspecific binding of the
antibodies and cellular autofluorescence are accounted for. For each
condition 104 cells were measured.
Results similar to those presented later in Figure 2 were
obtained when monoclonal antibody VC5, which is the same isotype as the
irrelevant control antibody (MOPC21), or a different secondary antibody, Alexa488-goat antimouse (Molecular Probes), was
used (data not shown).
Immunofluorescence
Where indicated, cells were loaded with either quin2/AM or the
calcineurin inhibitory peptide CN412. The cells were plated for 5 minutes at 37°C on coverslip dishes that had been previously coated
for 1 hour with a solution of 100 µg/mL fibronectin (GIBCO BRL).
Cells were then stimulated for 5 minutes with 10 nmol/L fMLP, fixed,
and permeabilized simultaneously by incubation with 6.6%
paraformaldehyde/0.05% gluteraldehyde/0.25 mg/mL saponin in PBS for 2 minutes at 37°C. Nonspecific binding sites were blocked for 10 minutes with PBS containing 10% calf serum (blocking buffer). To
visualize F-actin, samples were stained with 1 U/5 µL FITC-conjugated phalloidin (Molecular Probes). For indirect immunofluorescence, cells
were incubated with primary antibody for at least 1 hour at room
temperature, washed extensively, then incubated with the appropriate
secondary for an hour. Images of fluorescent-labeled cells were
obtained using a Leica DMIRB (Leica Mikroscopie und Systeme GmbH,
Germany) equipped with a 63 × 1.32 numerical
aperture objective. Images were acquired with a Princeton
Instruments (Princeton, NJ) cooled CCD camera driven by
Image-1/MetaMorph Imaging System software (Universal Imaging
Corporation, PA). Alternatively, cells were visualized on a Bio-Rad
MRC600 laser scanning confocal microscope (Bio-Rad Microscience,
Cambridge, MA) and a z-stack was obtained. Maximum projection images
were produced using Image-1/MetaMorph Imaging System software.
Labeling polymorphonuclear neutrophils with Cy3-VC5 and
C6-NBD-gal
A nonfunction blocking monoclonal anti-5 antibody (VC5,
Pharmingen) was directly conjugated to the fluorophore Cy3 (Amersham) according to the manufacturer's instructions. To block nonspecific and
Fc receptor binding sites, PMNs were first incubated in the presence of
25 µg/mL of an irrelevant isotype-matched antibody (MOPC21, Sigma)
for 10 minutes on ice. Surface-expressed 5 were then labeled by
incubation with 10 µg/mL Cy3-VC5 in the continuing presence of MOPC21
for an additional 30 minutes on ice. For some experiments, during the
last 30 seconds of incubation with the Cy3-VC5 and MOPC21 antibodies,
the plasma membrane of PMNs was labeled with C6-NBD-gal as
described below.
N-([6-[7-nitrobenz-2-oxa-1,3-diazol-4-yl]amino] hexanoyl)
sphingosyl phosphocholine (C6-NBD-gal) was prepared as
described previously.29 C6-NBD-gal/lipid
vesicles (100 µmol/L total lipid) were prepared by injecting an
ethanolic solution of a 1:1 mixture of C6-NBD-gal and
dioleylphosphatidylcholine (DOPC; 2.5 mmol/L total lipid; Avanti Polar
Lipids, Inc, Albaster, AL) into 150 mmol/L NaCl, 20 mmol/L HEPES, pH
7.4. The plasma membrane of PMNs was labeled by resuspending the cells
into a 1:30 dilution of the stock C6-NBD-gal/lipid vesicle
solution in incubation buffer. PMNs were then incubated for 30 seconds
at room temperature, washed once with incubation buffer, then placed on
ice until ready for use. For some experiments, C6-NBD-gal
was removed from the plasma membrane by incubating the cells in the
presence of serum-containing medium (back exchange
medium).30
When simultaneous DIC and fluorescence images were required, time-lapse
microscopy of C6-NBD-gal-labeled cells was accomplished with a Zeiss LSM510 laser scanning confocal microscope (Carl Zeiss Inc,
Jena, Germany) with the pinhole opened to maximize the depth of field.
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Results |
Effect of function-blocking anti-integrin antibodies on
neutrophil motility
As previously shown,4,5 neutrophil motility on
fibronectin is inhibited when [Ca++]i
transients are suppressed by the intracellular calcium buffer quin2. In
a previous study, time-lapse video microscopy revealed that cells
loaded with quin2 are able to send out pseudopods, but they are unable
to detach their uropods from fibronectin-coated surfaces.2
To determine whether 51 integrin is responsible for
[Ca++]i-sensitive motility of PMNs on
fibronectin, we measured the ability of function-blocking antibodies to
restore motility to [Ca++]i-buffered cells.
When [Ca++]i-buffered cells were incubated in
a blocking polyclonal antibody to 51, motility was restored to
near control levels (Figure
1A).
Incubation with a polyclonal antibody to v3 had no effect on this
motility. To further characterize the integrin responsible for
[Ca++]i-sensitive motility on fibronectin, we
measured the ability of function-blocking monoclonal antibodies to
restore motility to [Ca++]i-buffered cells
(Figure 1B). Monoclonal antibodies to 5 and 1 or the 51
complex were able to restore motility in
[Ca++]i-buffered cells. Both control and
[Ca++]i-buffered PMNs that are treated with
these antibodies exhibit the typical amoeboid-like motility displayed
by untreated control cells. 2 integrins are the most abundant
integrins on neutrophils, but antibodies to 2 integrins do not
restore motility to [Ca++]i-buffered
neutrophils on fibronectin, indicating that they are not the
[Ca++]i-sensitive fibronectin-binding
integrin (data not shown). In adhesion assays, monoclonal antibodies
against either 51 or 2 integrins alone only partially inhibit
adhesion of PMNs to fibronectin; addition of both anti-51 and
anti-2 antibodies is necessary to completely abrogate adhesion (not
shown). Thus, 2 integrins are presumably responsible for adhesion
and motility when 51 integrins are blocked, but the 2
attachments do not exhibit
[Ca++]i-sensitivity.
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| Fig 1.
Motility restoration of calcium-buffered neutrophils on
fibronectin with polyclonal antibodies.
(A) Where indicated, cells were
[Ca++]i-buffered by a 40 minutes' incubation
in 50 µmol/L Quin2/AM. The cells were then incubated with a 1:2000
dilution of the indicated antisera, rinsed, plated on a
fibronectin-coated coverslip dish, and stimulated with 10 nmol/L fMLP
in the continued presence of antisera. (B) Motility restoration of
calcium-buffered neutrophils on fibronectin with monoclonal antibodies.
Where indicated, neutrophils were
[Ca++]i-buffered with Quin2/AM as in (A),
incubated with a 5 µg/mL solution of the indicated monoclonal IgG,
rinsed, plated on a fibronectin-coated coverslip dish, and stimulated
with 10 nmol/L fMLP in the continued presence of antibody. Cells able
to move more than 7 µm in 200 seconds were considered motile. In each
case, more than 150 cells were assayed. The data shown are mean values ± SEM.
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| Fig 2.
Identification of integrins by flow cytometry.
Neutrophils were incubated in 5 µg/mL of the monoclonal antibodies
JB55 (5) (A), CLB-705 (51) (B), or JB1a (1) (C) in the
presence of fMLP (solid lines). For comparison, cells were incubated
with an irrelevant control antibody MOPC21 (short dashes) or with the
monoclonal antibody against 2 integrin, IB4 (long dashes). The cells
were rinsed with PBS and then incubated in a fluorescein-conjugated
secondary antibody. The cells were fixed with paraformaldehyde before
flow cytometry. Intensity histograms for each of the monoclonal
antibodies are shown. For each condition, 1 × 104
cells were measured.
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Because the 1 integrin subunit can also form heterodimers with the
6 integrin chain, we measured the ability of blocking antibodies to
6 integrin to restore neutrophil motility on fibronectin. [Ca++]i-buffered cells that were incubated in
antibody to 6 integrin showed no increase in motility (data not
shown). Similarly, the monoclonal antibody to v3 (LM609), which
has been shown to restore [Ca++]i-sensitive
motility on vitronectin,31 was not able to restore motility
to these cells on fibronectin. These results indicate that the
fibronectin-binding integrin 51 is responsible for [Ca++]i-sensitive motility of PMNs on
fibronectin substrates.
51 expression on polymorphonuclear neutrophils
We used flow cytometry to confirm that 51 integrin is indeed
expressed in neutrophils. We performed binding assays on each of the
antibodies used in these studies to determine the concentration of
antibodies necessary to saturate all binding sites (data
not shown), thereby allowing us to make estimates of the relative concentrations of 51 integrin compared with other integrins. Integrins on PMNs were labeled with saturating concentrations of
primary antibodies, followed by fluorescently conjugated secondary antibody (Figure 2). PMNs express significant amounts of 1 integrin subunits (Figure 2C). The fluorescence intensity seen using antibodies to 5 (A) or 51 (B) integrin is slightly less than 50% of that associated with the 1 integrin subunit alone. This is consistent with the association of other chains with 1. Because it has been
shown that 61 integrin is also present in
neutrophils,15,32 we used antibodies to the 6
integrin subunit (CLB-701) to determine what concentration of 1 was
due to this complex. The fluorescence intensity of cells labeled with
anti-6 integrin antibody accounts for approximately 60% of the 1
concentration (data not shown). Because the 51 integrin
heterodimer accounts for 40% to 45% of the 1 integrin subunit and
61 accounts for approximately 60%, 51 and 61 appear
to comprise the major 1 containing integrins in neutrophils. The
fluorescence intensity associated with 51 integrins is
approximately 10% of that associated with 2 integrins (compare
solid with long-dashed lines in Figure 2B). Neutrophils express
approximately 4 to 5 × 105 2
integrins,33 so on the basis of the relative fluorescence intensities, we can estimate that neutrophils express approximately 4 to 5 × 104 51 integrins. It should be noted
that this calculated expression level of 51 integrin is only a
rough estimate because it was obtained using indirect
immunofluorescence. Nevertheless, these results show that PMNs have
detectable amounts of 51 and that 51 is at much lower
expression levels than the 2 integrins.
Immunofluorescence of 5 and 1 in polarized polymorphonuclear
neutrophils
To determine the localization of integrins in fMLP-stimulated
neutrophils, we compared the distribution of 5 integrins with that
of the predominant integrins expressed on PMNs, the 2 integrins. Localization of the 5 integrin was attained by labeling fixed and
permeabilized cells with a monoclonal antibody to the extracellular domain of 5, followed by visualization with a fluorophore-conjugated secondary antibody. With this procedure, we found that this integrin is
localized toward the leading edge of the cells (Figure
3C). Much of the leading edge staining is
lost when immunofluorescence is performed on nonpermeabilized cells,
possibly because tight attachment at the front of the cells excludes
the antibody. The punctate fluorescence seen throughout the cell is
also lost when immunofluorescence is performed on nonpermeabilized
cells, and this also may be because antibody is excluded from the lower
adherent surface or because the integrin is located in intracellular
vesicles (see below). In contrast to the 5 integrin, the 2
integrins are generally found in the back third of both permeabilized
(Figure 3D) and nonpermeabilized cells (not shown), and this integrin is never seen in a band at the leading edge. The image shown in Figure
3D was obtained with MAB1962 (Chemicon) and differs somewhat from that
obtained with other antibodies against 2 (eg, IB4 and MAB44). With
these latter antibodies, much of the fluorescence in permeabilized
cells is derived from 2 integrins in intracellular vesicles, making
it difficult to discern the surface distribution of this integrin. All
the antibodies tested consistently gave a distribution for 2 that
was toward the back third of the cells.
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| Fig 3.
5 integrin, compared with 2, is enhanced toward the
front of polarized PMNs.
PMNs were plated on fibronectin, stimulated with 10 nmol/L fMLP, and
then simultaneously fixed and permeabilized with 6.6%
paraformaldehyde/0.05% gluteraldehyde in PBS containing 0.25 mg/mL
saponin. Samples were incubated with 5 µg/mL of either a monoclonal
antibody to the extracellular domain of 5 integrin (VC5, panel C),
or a monoclonal antibody to the extracellular domain of 2 integrin
(MAB1962, panel D). The samples were rinsed with PBS and then incubated
with a TRITC-conjugated secondary antibody. The leading edge of the
cells can be determined morphologically from differential interference
contrast (DIC) images (A, B). Bar = 10 µm.
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As seen in Figure 4A and B,
[Ca++]i-buffered PMNs have a morphology that
is quite distinct from the control cells shown in Figures 3A and B. After calcium buffering with quin2/AM, time-lapse video microscopy
showed that PMNs continue to extend pseudopodia, but they are
unable to detach at the rear, and consequently they become elongated (data not shown and Marks and Maxfield2). In
these cells, there is a marked decrease in punctate 5 staining
throughout the cell (compare Figure 4C with Figure 3C). In contrast to
untreated cells (Figure 3C), most of the 5 is found accumulated in
clusters at the rear of [Ca++]i-buffered
cells (Figure 4C); the localization of the 2 integrins toward the
rear of the cells is unaffected by
[Ca++]i-buffering (compare Figure 3D with
4D). From 2 different days experiments, an average of 81% of
[Ca++]i-buffered cells (n = 86) became
elongated and 86% of these elongated cells showed an accumulation of
5 in the uropod.
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| Fig 4.
[Ca++]i-buffering
affects the localization 5 but
not 2 integrin in PMNs.
PMNs were loaded with quin2/AM as described in "Materials and
methods," then plated onto fibronectin, stimulated with fMLP, and
fixed and permeabilized as for Figure 3. 5 (C) and 2 (D)
integrins were visualized by indirect immunofluorescence with
monoclonal antibodies and a TRITC-conjugated secondary. DIC images are
shown in panels A and B. Bar = 10 µm.
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When a monoclonal antibody to 1 is used, we found that the protein
is also found toward the leading edge, but there is additional staining
throughout the cell (not shown) presumably because of 1 integrins
associated with 6 subunits. As with the 5 integrin, calcium-buffering of neutrophils caused a significant amount of the
1 integrin to be found in clusters toward the rear of the cell (not
shown). To avoid the contribution of 61 integrin, all subsequent
experiments were performed using an anti-5 antibody.
It has been shown previously that calcineurin inhibition
causes cells to become stuck on vitronectin but not on
fibronectin.7 We have shown that calcium transients act
through the calcium-dependent phosphatase calcineurin to break up
v3 integrin clusters in cells on vitronectin.6 We
introduced the calcineurin inhibitory peptide CN412 into neutrophils
using an osmotic shock technique7 and tested whether
calcineurin inhibition has any effect on 51 localization in cells
on fibronectin. To demonstrate that we had successfully introduced the
peptide into the cells, we verified that migration on vitronectin was
inhibited (data not shown). When cells are plated onto fibronectin, the
localization of 5 integrin is unaffected by inhibition of
calcineurin (not shown). Thus, in contrast to the effects on v3
distribution for cells migrating on vitronectin, there is no effect of
calcineurin inhibition on the distribution of 51 for cells
migrating on fibronectin.
Immunolocalization of actin-associated proteins
Talin and -actinin are proteins that link integrins to
the actin cytoskeleton.34-36 We have shown previously that
both talin and -actinin colocalize with integrin clusters at the
rear of cells that have been [Ca++]i-buffered
on vitronectin.6 Here we used monoclonal antibodies to both
talin and -actinin to determine whether calcium buffering causes a
redistribution of talin and -actinin to the rear of cells attempting
to migrate on fibronectin (Figure 5). Both
talin (Figure 5C) and -actinin (data not shown) are found
colocalized with F-actin (Figure 5B) near the leading edge of motile
cells on fibronectin, but in cells that are
[Ca++]i-buffered, F-actin and talin are found
in the rear of the cells, as well as in the leading lamella when one
exists (Figure 5D and F). Inhibition of calcineurin in cells crawling
on fibronectin had no effect on the localization of either F-actin or
talin (data not shown). These findings suggest the presence of
transient adhesion complexes in cells on fibronectin that require
elevations in free calcium to be disassembled. Unlike adhesion
complexes in cells on vitronectin, disassembly of adhesion complexes on
fibronectin is apparently independent of calcineurin.
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| Fig 5.
[Ca++]i-buffering
causes a redistribution of
F-actin and talin in PMNs.
PMNs were either loaded with quin2/AM (D-F) or not (A-C), then prepared
for immunofluorescence as described. Samples were incubated in 5 µg/mL of a monoclonal antibody to talin, rinsed, and then stained
with a TRITC-conjugated secondary antibody and FITC-conjugated
phalloidin to visualize F-actin. In control cells (A-C), talin (C) is
found colocalized with F-actin (B) predominately at the leading edge of
motile cells. In contrast, when cells are
[Ca++]i-buffered both talin (F) and F-actin
(E) are found at the rear of cells as well as in the leading lamella if
one exists. DIC images are shown (A, D). Bar = 10 µm.
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Confocal microscopy of 5 integrin
To determine whether the punctate 51 integrin staining that we
observed via wide-field fluorescence microscopy (Figure 3) was due to
integrin localized in intracellular vesicles or in focal contacts on
the lower adherent surface of neutrophils, we used confocal microscopy
to distinguish between intracellular integrins and those on the
surface. In motile cells (Figure 6A-D), a
large portion of the anti-5 staining is seen throughout the cytoplasm of the cell and at the leading edge. The cytoplasmic 5 is
seen in a large number of intracellular vesicles and in a perinuclear
compartment. To show that the vesicles and this compartment are
intracellular, Figure 6C shows a single horizontal plane through the
cells shown in Figure 6A; this plane corresponds to the plane marked by
the arrows in Figure 6B and is clearly above the adherent surface of
the cell. Figure 6D shows a single x-z slice through one of the cells
along the axis indicated by arrows in Figure 6A.
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| Fig 6.
Confocal imaging of 5
integrin in control and
[Ca++]i-buffered
PMNs plated on fibronectin.
PMNs were either loaded with quin2/AM (E-H) or not (A-D), then prepared
for immunofluorescence as in Figure 3. Samples were incubated with 10 µg/mL of a monoclonal antibody to the extracellular domain of 5
integrin, rinsed with PBS, and then incubated with a TRITC-conjugated
secondary antibody. The samples were viewed using a Bio-Rad MRC 600 laser scanning confocal microscope and vertical sections were obtained.
x-y (A, E) and x-z (B, F) projections of both control (A-D) and
quin2-buffered (E-H) cells are shown. Panels C and G show the
localization of 5 at a single x-y slice at the depth indicated by
the arrows in panels B and F, respectively. Panels D and H show a
single x-z slice along the axis of the cells indicated by the arrows in
panels A and E, respectively. Bar = 10 µm.
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Cells that have been calcium buffered with quin2 show significantly
less 5 in intracellular vesicles inside the cell (Figure 6E-H).
There is a large amount of 5 on the lower surface of the cell,
accumulated in the pronounced uropod. There is still some 5 found in
intracellular vesicles and in the perinuclear compartment, but it is
markedly less than that seen in control cells. This depletion of 5
integrin from intracellular vesicles in calcium-buffered cells could be
due to an inhibition of either normal endocytic trafficking or 5
integrin release from integrin clusters. The latter interpretation is
supported by the finding that Ca++ buffering has no effect
on the rates of endocytosis and recycling of bulk membrane in
neutrophils (L.M.P. and F.R.M., unpublished results).
Intracellular localization of the 5 integrin subunit
The localization of 51 in intracellular vesicles suggests that
this integrin may be in endocytic compartments and thus may be recycled
during cell migration. Many cell surface proteins, such as the LDL and
transferrin receptors, as well as bulk lipid membrane, recycle along a
well-characterized pathway.37 In CHO cells that have been
transfected with the human transferrin receptor,38 fluorescently labeled transferrin has been used to characterize the
endocytic recycling pathway of the transferrin
receptor.29,39 After endocytosis but before exiting the
cell, the transferrin receptor accumulates in a pericentriolar ERC that
is separate and distinguishable from sorting endosomes.40
Together, sorting endosomes and the ERC comprise the early endosome
system. It has been shown previously that the fluorescent lipid analog,
C6-NBD-gal, as well as several other lipid analogues,
follow an endocytic recycling pathway that is indistinguishable from
that followed by the transferrin receptor in CHO and other
cells.29 Because PMNs do not express significant amounts of
the transferrin receptor, we used C6-NBD-gal as a marker of
the ERC in PMNs and looked for colocalization with the 5 subunit.
When PMNs are labeled with C6-NBD-gal and maintained on
ice, the C6-NBD-gal labeling is restricted to the plasma
membrane (Figure 7D). After warming the
cells to 37°C, some of the C6-NBD-gal is endocytosed
from the plasma membrane and is seen accumulated in an intracellular
perinuclear compartment (Figure 7F and Figure 8C). In Figure 7, panels F and H,
C6-NBD-gal was removed from the plasma membrane by
incubation in back exchange medium. Immediately after the plasma
membrane-associated C6-NBD-gal was removed, arrays of
vesicles (small arrows, Figure 7F) are seen that appear to either be
emanating from or converging into the central compartment. When PMNs
are incubated for longer times (30-60 minutes) in back exchange medium,
C6-NBD-gal can be chased out of the perinuclear compartment
and out of the cell (Figure 7H), confirming that C6-NBD-gal indeed labels a recycling compartment in these cells. The kinetics with
which C6-NBD-gal leaves PMNs is similar to that of
C6-NBD-gal exocytosis from CHO cells (data not shown),
suggesting that the C6-NBD-gal-labeled compartments in
each of these cells are analogous.
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| Fig 7.
C6-NBD-gal on
the surface of PMNs is
endocytosed and trafficked through
an endocytic recycling compartment
(ERC) before being transported
back out to the cell
surface.
PMNs were labeled with the fluorescent lipid analogue
C6-NBD-gal (C-F) or with a Cy3-conjugated,
nonfunction-blocking monoclonal antibody to 5 (Cy3-VC5; A, B), then
maintained at 4°C (A-D) or warmed to 37°C for 30 minutes (E-H).
After this warm-up, cells were incubated for an additional 2 minutes
(E, F) or 30 minutes (G, H) in back exchange medium. Cy3-VC5 and
C6-NBD-gal are initially found on the plasma membrane
(A-D). After a 30-minute incubation at 37°C, both Cy3-VC5 and
C6-NBD-gal accumulate in a central compartment (see Figure
8). Two minutes after back exchange of C6-NBD-gal from the
plasma membrane, a central compartment is clearly visible in the cells
(large arrows, panel F) and in some cases
C6-NBD-gal-labeled vesicles (small arrows, panel F) appear
to be emanating from the central compartment. By 30 minutes after
warm-up, almost all of the C6-NBD-gal has been returned to
the plasma membrane and been back exchanged into the medium (H). DIC
images are shown in (A, C, E, and G). Bar = 10 µm.
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| Fig 8.
5 integrin colocalizes
with a lipid marker of
the ERC.
PMNs were incubated with Cy3-VC5 and MOPC21 for 30 minutes on ice, then
labeled with C6-NBD-gal. Cells were washed well, incubated
at 37°C for 30 minutes, then plated onto fibronectin-coated
coverslip dishes. PMNs were incubated in the presence (D-F) or absence
(A-C) of fMLP before fixation with 2% paraformaldehyde. Cy3-VC5 (B, E)
is seen on the plasma membrane, as well as accumulated in a centrally
located cluster inside the cells. The intracellular clusters of 5
colocalize with a compartment labeled with C6-NBD-gal (C,
F) in both unstimulated (A-C) and stimulated (D-F) cells. DIC images
are shown in (A, D). Bar = 10 µm.
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As with the C6-NBD-gal labeling, 5 labeled with a
directly conjugated nonblocking antibody (Cy3-VC5) is restricted to the plasma membrane for PMNs maintained on ice (Figure 7B). After a warm-up
to 37°C, 5 is found in a perinuclear compartment that colocalizes with that labeled by C6-NBD-gal in both resting
(Figure 8A-C) and stimulated (Figure 8D-F) cells. The colocalization of the 5 integrin subunit with a bulk membrane marker in PMNs
demonstrates that integrins are found in an ERC. In apparent contrast
to Figure 3, 5 integrin is absent from the leading edge of the
polarized cell shown in Figure 8E. For this experiment, only those
51 initially expressed on the surface of PMNs were labeled.
Excess antibody was removed by washing the cells before stimulating the cells to migrate. Thus, integrins that are newly delivered to the cell
surface after stimulation would not be labeled by the directly
conjugated antibody. That the front of the cell is not labeled is
consistent with our model because that is where we would expect new
integrins to be delivered (see Figure 10).
Localization and orientation of the endocytic recycling compartment
during polymorphonuclear neutrophils migration
Because it has been reported that the nucleus and MTOC of migrating
PMNs reorient as the cell moves and because in many cell types the ERC
is closely associated with the MTOC, we used time-lapse microscopy to
determine the localization of the ERC during PMN migration. The plasma
membrane and ERC of PMNs were labeled with C6-NBD-gal as
above, then C6-NBD-gal was back exchanged from the plasma
membrane just before plating and stimulating the cells. Immediately
after stimulation, DIC and fluorescence time-lapse images were
acquired. The outline of the cell, as well as the border between the
cell body and the leading lamella, shown in the right panels in Figure
9, were
determined from the DIC images on the left. The large active leading
lamella of the PMN appears slightly out-of-focus and irregular by DIC.
The appearance of leading lamellae of PMNs is unlike the flat and
smooth appearance of leading lamellae of other cell types, yet they are
still clearly distinguishable from the sharply focused
granule-containing cell bodies. Time-lapse fluorescence microscopy of
motile cells revealed that the ERC is almost always (43/45 cells)
located just behind the leading lamellae in migrating PMNs. In all but
1 cell (12/13) that made a dramatic change in direction during the
experiment, the ERC reoriented as the cell migrated so that it retained
its position just behind the lamella (Figure 9). This asymmetric
localization of the ERC is in contrast to the central localization
found in resting cells (see Figure 7F), and it in effect imparts a
polarity to the cell's recycling mechanism that may play an important
role in cell migration (see "Discussion").
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| Fig 9.
The ERC is localized
just behind the leading lamella
of motile PMNs and reorients
to retain this position as
the cells move.
The plasma membrane of PMNs was labeled with C6-NBD-gal as
described in "Materials and methods." The ERC was labeled by
incubating C6-NBD-gal-labeled PMNs for 10 minutes at
37°C. To remove C6-NBD-gal from the plasma membrane,
yet retain C6-NBD-gal labeling of the recycling
compartment, cells were incubated for an additional 10 minutes on ice
in back exchange medium. PMNs were plated onto fibronectin-coated
coverslip dishes, stimulated with fMLP, then imaged with a Zeiss LSM510
confocal microscope. DIC and fluorescence images were acquired
simultaneously at the indicated time points (numbers represent time in
seconds). Outlines of the cells and the boundary between the cell body
and the lamella were obtained from the DIC images, then transferred to
the fluorescence images. Bar = 10 µm.
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| Fig 10.
Diagram of regulation of
PMN motility on fibronectin.
In panel A, 51 integrins (cup shapes on stems) at the leading
edge form tight attachments to the fibronectin substrate (filled ovals
on stems). During migration, PMNs move forward over the attachment
sites while forming new attachments toward the front and releasing old
ones toward the rear. The released integrins near the rear are
internalized into endocytic vesicles (open circles inside cell) then
delivered via the ERC (gray freeform inside cell) toward the front of
the cell. Eventual delivery of integrins to attachment sites occurs by
an unknown mechanism. When intracellular Ca++ is buffered
(panel B), old attachments cannot be released causing 51
integrins to accumulate at the back of the cell and become depleted
from endocytic compartments.
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Abstract
Introduction