University of Massachusetts Medical School, Department of Physiology, 377 Plantation, Room 327, Worcester, MA 01605, USA
* Author for correspondence (e-mail: yuli.wang{at}umassmed.edu )
Accepted 13 November 2001
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Summary |
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Key words: Mechanical sensing, Cytoskeleton, GTPase, Rac1, Lysophosphatidic acid, Phosphotyrosine
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Introduction |
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Many aspects of Fc-receptor-mediated phagocytosis resemble events at the
leading edge of a migrating cell. Both processes are actin-mediated and both
involve adhesion, receptor clustering, tyrosine phosphorylation,
lamellipodial/pseudopodial extension, and force generation
(Aderem and Underhill, 1999;
Sheetz et al., 1998
). For cell
migration, it is becoming increasingly clear that both chemical and physical
parameters of the substrate play an important role
(Sheetz et al., 1998
). For
example, transient mechanical stimuli can activate cell migration and neurite
growth (Verkhovsky et al.,
1999
; Bray, 1984
),
possibly through the reorganization of the actin cytoskeleton and focal
adhesions (Heidemann et al.,
1999
; Riveline et al.,
2001
). In addition, substrate flexibility was found to affect both
the rate and direction of fibroblast migration
(Pelham and Wang, 1997
;
Lo et al., 2000
). The
similarities between lamellipodia extending on a substrate and phagocytic cups
forming around the target raises the possibility that mechanical signaling may
play an equally important role in phagocytosis.
To test this hypothesis, we have fed macrophages with polyacrylamide microbeads of identical surfaces but different stiffness. Our results provide convincing evidence that target rigidity indeed plays a determining role in phagocytosis. We show that adhesion, cortical cytoskeleton, and protein phosphorylation respond differently to rigidity signals and that the mechanosensitivity is regulated by lysophosphatidic acid (LPA) and the small GTP-binding protein Rac1.
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Materials and Methods |
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Bovine serum albumin (BSA) was covalently coupled to the polyacrylamide beads or sheets after activating the surface with 1-ethyl-3-(3-dimethylaminopropyl) (EDC; Pierce, Rockford, IL), followed by incubation with affinity purified rabbit anti-BSA IgG (Cappel, Aurora, OH or Sigma, St Louis, MO) for 1 hour at 24°C. Large particles (>6 µm) were removed by centrifugation in a microfuge at 300 g for 3 minutes. The remaining beads, 1-6 µm in diameter, were used for the experiments. In some experiments, beads were further incubated with 3 µM LPA (Avanti Polar Lipids, Alabaster, AL) in PBS for 1 hour at 24°C. The concentration of beads was determined before each phagocytosis experiment as follows: a 30 µl sample of opsonized beads was placed onto a slide, dispersed uniformly under a 22 mm circular coverslip, and allowed to settle for at least 30 minutes before counting. The concentration was calculated based on the average number of beads counted within five square areas of 200x200 pixels.
To determine the extent of antibody coating, opsonized beads were labeled
with fluorescent secondary antibodies for 1 hour at 24°C (Alexa 546
anti-rabbit IgG, Molecular Probes, Eugene, OR), and the fluorescence intensity
of single beads measured with a cooled CCD camera coupled to a microscope
(Fig. 1a). In some experiments,
the results of microscopy photometry were verified with flow cytometry. The
density of surface coating on sheets was tested as previously described
(Fig. 1b,c)
(Lo et al., 2000), using 1
µm Fluorsbrite carboxylate beads coated with antibodies against rabbit IgG
(Polysciences, Warrington, PA).
|
Preparation of macrophages and induction of phagocytosis
Bone-marrow-derived macrophages from 4-6-week-old C3H/HeJ mice were
prepared essentially as described previously
(Swanson, 1989), using
recombinant huCSF-1 (R&D Systems, Minneapolis, MN) to induce the
differentiation. Equal numbers (
1x106 cells per 35 mm
diameter area) of 5-8 day old cells, in growth medium without CSF-1, were
plated onto coverslips 24 hours before the experiments. The cells were rinsed
and acclimated for 10 minutes at 37°C in PBS. Equal numbers of beads
(
1x107 beads per 35 mm diameter area) were then
presented to the cells and incubated at 37°C for 45 minutes for the uptake
assays, or 10 minutes for immunofluorescence staining. Cells were then fixed
as previously described (Pelham and Wang,
1997
), and the ingested beads counted with a combination of phase
and fluorescence optics. Completely engulfed beads appeared as bright green
particles in fluorescence optics (Fig.
2b), but were phasedense and barely detectable in phase optics
(Fig. 2a;
Fig. 3c). This relationship
between phase morphology and engulfment was confirmed with 3D reconstruction
of optical sections of cells stained with fluorescent phalloidin
(Fig. 2c). The phagocytic index
is defined as the average number of beads engulfed per cell after 45 minutes
of incubation.
|
|
For the experiments of frustrated phagocytosis, cells in PBS were allowed to interact with opsonized polyacrylamide sheets for 45 minutes before fixation and staining with rhodamine phalloidin (Molecular Probes, Eugene, OH). Those with lamella extending in a radially symmetric fashion were counted as positive (Fig. 3e).
Fluorescent staining, microinjection and microscopy
Fixed cells were blocked with 1% BSA and stained for 1 hour at 37°C
with rhodamine phalloidin for filamentous actin (Molecular Probes), with a
monoclonal antibody pTyr-clone 4G10 for phosphotyrosine (1:100; Upstate
Biotechnology, Lake Placid, NY), or with a monoclonal antibody clone 349 for
paxillin (1:200; ICN, Costa Mesa, CA). Anti-mouse IgG coupled to the Alexa 546
fluorophore (Molecular Probes) was used at 1:1000 dilution as the secondary
antibody.
Constitutively active L61Rac1 and L63RhoA, and dominant negative
N17Rac1-GST were purchased from Cytoskeleton (Denver, CO), and resuspended and
stored as recommended by the manufacturer. C3 transferase was received as a
gift from S. Narumiya (Kyoto University, Kyoto, Japan) and used as described
previously (O'Connell et al.,
1999). The protein or carrier solution was microinjected as
previously described (Wang,
1994
), into murine-derived macrophages starved of m-CSF for 24
hours or into REF cells serum starved for 16 hours as controls. Rac1 and RhoA
proteins were injected at a needle concentration of 800 ug/ml, and C3 was
injected at a needle concentration of 200 ug/ml. The carrier solution
contained 7 mg/ml rhodamine dextran (70 kDa, lysine fixable; Molecular Probes)
as a marker. Following microinjection the cells were allowed to recover for 20
minutes in 37°C PBS and the phagocytosis experiments were carried out as
described above. The number of beads ingested by injected cells was counted
after fixation.
Cells were observed with a Zeiss Axiovert 10 or Axiovert TV100 microscope, equipped with a quartz halogen lamp and a 40x, NA 0.75 Plan-Neofluar objective lens. Images were recorded with a slow-scan cooled CCD camera (ST133 with CCD57 chip; Roper Scientific, Trenton, NJ) and processed for background subtraction. Optical sections of phalloidin images were processed with a custom feature detection program that highlights linear structures and outlines of phagosomes, and reconstructed into perspective views for stereo visualization.
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Results |
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As shown in Fig. 3a, macrophages showed a sixfold preference for the stiff over the soft opsonized beads. Naked beads or beads coated with BSA alone served as controls; both of which showed poor adhesion to the cell surface and a very low phagocytic index (<0.05). Although opsonized soft particles were ingested poorly, they were able to adhere to the surface of macrophages (Fig. 3f,g). In addition, no increase in the number of ingested soft beads was observed after an extended incubation of 2 hours, indicating that the difference between stiff and soft beads was not due to different rate of ingestion. Instead, the observations suggest that phagocytosis was inhibited downstream of receptor binding.
In a parallel experiment, we plated macrophages on polyacrylamide sheets of
different rigidity but opsonized with a similar density of IgG
(Fig. 1b,c). The cells spread
dramatically in an attempt to engulf the substrate
(North, 1968), a phenomenon
termed frustrated phagocytosis (Henson,
1971
; Rabinovitch and
DeStefano, 1973
) (Fig.
3e). The results again revealed a strong preference for the stiff
over the soft substrate (Fig.
3b,e,h).
To determine whether the binding of stiff or soft beads caused a global positive or negative response of phagocytosis, macrophages were incubated with a mixture of equal numbers of opsonized hard and soft beads, labeled with different colors of fluorescent dextran. Comparisons of phagocytic indices of hard versus soft beads showed no difference in the phagocytic uptake of either the hard or soft beads when presented in combination or individually, suggesting that target stiffness elicits local responses that determine the efficiency of phagocytosis.
Target rigidity affects the organization of actin filaments but not
paxillin or phosphotyrosine
Both actin cytoskeleton and adhesion structures are involved in
phagocytosis (Aderem and Underhill,
1999; May and Machesky,
2001
) and both are known to respond to mechanical stimuli in
fibroblasts (Heidemann et al.,
1999
; Riveline et al.,
2001
). Staining with rhodamine phalloidin showed that actin
filaments were concentrated beneath 85% of the stiff beads (n=100),
but only 11% of the soft beads (n=100;
Fig. 4a,b). This lack of actin
organization is probably responsible for the failure of cells to engulf soft
particles. However, staining of paxillin
(Fig. 4e,f) or vinculin (data
not shown), two proteins localized to the adhesive sites of Fc-mediated
phagocytic cups (Greenberg et al.,
1994
; Allen and Aderem,
1996
), showed that qualitatively both proteins became localized
beneath stiff or soft beads. Thus the initial recruitment of these proteins to
nascent phagocytic cups probably occurs upstream of actin assembly,
independent of the stiffness of the target.
|
It was previously reported that protein tyrosine phosphorylation increases
during both phagocytosis (Greenberg et
al., 1993) and mechanical stimulations
(Pelham and Wang, 1997
;
Schmidt et al., 1998
). This
response may play a role in mediating actin organization upon the binding of
stiff beads. However, both stiff (99%; n=100) and soft beads (98%;
n=100) induced a concentration of phosphotyrosine at the binding site
(Fig. 4c,d), suggesting that
tyrosine phosphorylation also lies upstream of the mechanosensitive mechanism.
However, it is possible that stiff beads may induce quantitatively a higher
extent of phosphorylation at steady state. It is also possible that
mechanosensing may involve differential tyrosine phosphorylation of a minor
protein, which cannot be discerned by the global staining.
The small GTPase Rac1 is involved in mechanosensing
The small GTPases are effectors of cytoskeletal organization and are known
to be involved in phagocytosis (May and
Machesky, 2001). To test the involvement of RhoA and Rac1 in
mechanosensing, we microinjected constitutively active L63RhoA and L61Rac1
into macrophages prior to the presentation of hard or soft beads.
Interestingly, constitutively active Rac1 not only created extensive ruffling
and lamellipodial activities as previously reported
(Allen et al., 1997
), but also
stimulated the ingestion of opsonized soft beads to a level similar to that
for stiff beads (Fig. 5a).
Moreover, microinjection of dominant negative N17Rac1 inhibited the uptake of
hard particles (Fig. 7a),
consistent with previous reports (Caron and
Hall, 1998
).
|
|
By contrast, microinjection of L63RhoA had no effect on the ingestion of
stiff or soft beads (Fig. 5a).
Similarly, microinjection of the C3 transferase, a specific inhibitor of Rho,
had no effect on phagocytosis (Fig.
7b), although it did cause the cells to spread out as described in
Bac1.2F5 cells (Allen et al.,
1997). Serum-starved REF cells did respond to L63RhoA by forming
stress fibers (Fig. 5b)
(Ridley and Hall, 1992
), and
to C3 transferase by rounding up (Fig.
7c) confirming the activity of these proteins. Together, these
results support the notion that Rac1, but not RhoA, plays an important role in
mechanosensing during Fc-receptor-mediated phagocytosis.
LPA stimulates phagocytosis of soft particles
To attempt to identify external factors that may modulate mechanosensing of
phagocytosis, we incubated opsonized beads with lysophosphatidic acid (LPA), a
natural serum component that functions as an activator of several signaling
pathways including the Rho GTPases (Ridley
and Hall, 1992; Moolenaar
1999
; Koh et al.,
1998
). LPA greatly stimulated the phagocytosis of opsonized soft
beads, to an extent similar to that of stiff particles
(Fig. 6a). Actin filaments
became concentrated beneath 85% of the soft beads (n=200;
Fig. 6b,c), similar to what was
found with stiff beads. However, incubation of stiff beads with LPA did not
induce further enhancement of phagocytosis, nor did cells engulf non-opsonized
soft beads coated with BSA and LPA (Fig.
6a). Thus, rather than activating an independent, indiscriminate
mechanism of phagocytosis, LPA probably elicits a signal normally stimulated
by the rigidity of the opsonized target.
|
It is possible that LPA stimulates phagocytosis of soft particles through
the activation of Rac1, as was suggested in some systems
(Moolenaar, 1999). Consistent
with this idea, dominant negative Rac1 abolished the ingestion of LPA-coated
soft particles (Fig. 7a), while
C3 had no effect (Fig. 7b).
However, we detected no global elevation of Rac1 activities following the
treatment with LPA alone (data not shown), suggesting that the stimulation of
Rac1 by LPA requires Fc binding and thus may be localized near phagocytic
sites.
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Discussion |
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Our results suggest that, at least at a qualitative level, the engagement
of Fc receptors with IgG is sufficient for activating tyrosine phosphorylation
and the reorganization of paxillin and vinculin at the binding site. However
soft beads were unable to stimulate the assembly of actin filaments required
for the formation and closure of phagosomes. This mechanosensing mechanism
probably involves a force-dependent probing step, which lies downstream of the
initial steps of ligand binding and tyrosine phosphorylation. Owing to the
difficulty in measuring phagosome-associated proteins, we cannot rule out the
possibility that stiff targets stimulate a further increase in tyrosine
phosphorylation and vinculin/paxillin organization through a feedback
mechanism, as was observed at focal adhesions of fibroblasts
(Pelham and Wang, 1997).
Previous studies have implicated the Rho-family GTPases in regulating
cortical actin reorganization during Fc-mediated phagocytosis
(Chimini and Chavier, 2000).
The effects of this family of proteins, including Rho, Rac and Cdc42, on the
actin cytoskeleton are well established in non-macrophage cells
(Nobes and Hall, 1999
;
Chrzanowska-Wodnicka and Burridge,
1996
; Amano et al.,
1996
). Although these proteins have also been localized to
phagocytic cups (Caron and Hall,
1998
), their exact role in phagocytosis is less clear. Experiments
with C3 transferase have yielded contradictory results as to the involvement
of RhoA in Fc-mediated phagocytosis, while other studies have indicated the
involvement of Rac1 in mediating pseudopod extension and particle engulfment
(Caron and Hall, 1998
;
Hackam et al., 1997
;
Massol et al., 1998
;
Cox et al., 1997
;
Castellano et al., 2000
). Our
results support the notion that Rac1, but not RhoA, is involved in Fc-receptor
mediated phagocytosis. In addition, we provide evidence that Rac1 mediates a
mechanosensing process that leads to the preferential ingestion of stiff
particles. In the simplest scenario, mechanical input from rigid particles
causes the activation of Rac1, which in turn activates the assembly of actin
filaments at phagocytic cups. There are three known pathways, involving the
PAK/LIM kinases, phosphoinositides, and the Arp2/3 complexes respectively,
through which activated Rac1 could induce actin polymerization. Components of
all three pathways have been localized to the phagocytic cup
(Dharmawardhane et al., 1999
;
May et al., 2000
;
Botelho et al., 2000
).
Our observation that LPA bypasses the mechanosensing mechanism is
intriguing, given the stimulation of Rho by LPA in many other systems and the
lack of involvement of RhoA in the present experiments. However, LPA may
activate alternative GTP binding proteins during phagocytosis. In the present
study, the effects of LPA were inhibited by dominant negative Rac1, suggesting
that Rac1, instead of RhoA, is the primary effector of the LPA. This
LPA-modulated mechanosensing mechanism is likely to have broad implications in
vivo. For example, bacteria, yeast, and other pathogens with cell walls are
usually more rigid than the surrounding tissue they invade
(Doyle and Marquis, 1994;
Thwaites and Mendelson, 1991
),
thus the sensitivity of macrophage to rigidity would promote a more specific
ingestion of these pathogens. Furthermore, at sites of wounding and pathogenic
invasion, an elevation of LPA levels
(Moolenaar, 1999
) would reduce
the mechanical dependence of Fc-mediated phagocytosis and lead to more
aggressive attacks of invading microbials irrespective of their rigidity.
Conversely, it is also possible that improper regulation of mechanical sensing
can lead to autoimmune diseases such as rheumatoid arthritis. Given the
stiffness of cartilage relative to other tissues
(Hayes and Mockros, 1971
), its
mechanical properties may play an important role during the progression of
this disease. Defining mechanosensing in phagocytosis could facilitate the
development of new clinical approaches that target the mechanosensing pathways
of the cell to either promote the recognition of pathogens or interfere with
autoimmune responses.
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Acknowledgments |
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