Rigidity of Circulating Lymphocytes Is
Primarily Conferred by Vimentin
Intermediate Filaments
This information is current as
of June 18, 2017.
Martin J. Brown, John A. Hallam, Emma Colucci-Guyon
and Stephen Shaw
J Immunol 2001; 166:6640-6646; ;
doi: 10.4049/jimmunol.166.11.6640
http://www.jimmunol.org/content/166/11/6640
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References
Rigidity of Circulating Lymphocytes Is Primarily Conferred by
Vimentin Intermediate Filaments
Martin J. Brown,* John A. Hallam,* Emma Colucci-Guyon,† and Stephen Shaw1*
Lymphocytes need rigidity while in circulation, but must abruptly become deformable to undergo transmigration into tissue.
Previously, the control of leukocyte deformability has been attributed to microfilaments or microtubules, but the present studies
demonstrate the greater importance of vimentin intermediate filaments (IFs). In circulating T lymphocytes, IFs form a distinctive
spherical cage that undergoes a rapid condensation into a juxtanuclear aggregate during chemokine-induced polarization. Measurements of the resistance of peripheral blood T lymphocytes to global deformation demonstrate that their rigidity is primarily
dependent on intact vimentin filaments. Microtubules, in contrast, are not sufficient to maintain rigidity. Thus, vimentin IFs are
a primary source of structural support in circulating human lymphocytes, and their regulated collapse is likely to be an essential
element in chemokine-induced transendothelial migration. The Journal of Immunology, 2001, 166: 6640 – 6646.
*Experimental Immunology Branch, National Cancer Institute, National Institutes of
Health, Bethesda, MD 20892; and †Unité de Biologie du Développement, Institut
Pasteur, Centre National de la Recherche Scientifique Unité de Recherche Associée,
Paris, France
Received for publication June 9, 2000. Accepted for publication March 27, 2001.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
Address correspondence and reprint requests to Dr. Stephen Shaw, Human Immunology Section, Experimental Immunology Branch, National Cancer Institute, National Institutes of Health, Building 10, Room 4B36, 10 Center Drive MSC 1360,
Bethesda, MD 20892. E-mail address: [email protected]
2
Abbreviations used in this paper: F-actin, filamentous actin; DS, donkey serum; IF,
intermediate filament; KO, knockout; MTOC, microtubule organizing center; PBT,
peripheral blood T lymphocyte; PFA, paraformaldehyde; PLL, poly(L-lysine); SDF1␣, stromal cell-derived factor-1␣; WT, wild type.
Copyright © 2001 by The American Association of Immunologists
element in determining the rigidity of circulating lymphocytes, and
thus propose that their collapse during polarization is necessary to
achieve the increased cell deformability required for transendothelial migration.
Materials and Methods
Purification of CD4⫹ human T lymphocytes
PBMC were obtained from buffy coats derived from healthy donors by
centrifugation through lymphocyte separation medium (ICN, Aurora, OH).
Enriched populations of resting, CD4⫹ PBT were isolated from PBMC by
immunomagnetic negative selection, essentially as described previously
(14). The resulting resting CD4⫹ T cells (⬎95% purity) were suspended in
medium (HBSS, without phenol red, containing 10 mM HEPES and 0.2%
BSA) and either used immediately or held in suspension by rotation at 4°C
for up to 24 h. Cells were placed in polypropylene microcentrifuge tubes
at 107/ml and prewarmed to 37°C on a rocking platform for at least 1 h
before use in experiments.
Mouse splenic T lymphocyte cultures
The vimentin knockout (KO) mouse (Vim1 mutation) has been previously
described (15). Control 129/Sv mice were generously provided by Linda
Lowe (National Cancer Institute). All animals were treated according to
National Institutes of Health guidelines. Mice were sacrificed at 6 –10 wk
of age, and spleens were removed. Single cell suspensions were treated
with ACK lysing buffer and washed. Bulk cultures of mouse spleen cells
were initiated at a density of 2 ⫻ 106 cells/ml in RPMI 1640 (Life Technologies, Grand Island, NY) supplemented with 10% heat-inactivated FBS
(Summit Biotechnology, Ft. Collins, CO), MEM nonessential amino acids,
20 mM HEPES buffer, 1 mM pyruvate, 1⫻ penicillin/streptomycin (all
from Life Technologies), 50 ␮M 2-ME (Sigma, St. Louis, MO), 20 U/ml
human rIL-2 (Hoffmann-LaRoche, Nutley, NJ), 10% rat T-STIM (Becton
Dickinson Labware, Mountain View, CA), and 5 ␮g/ml Con A (Sigma).
After 40 h of culture at 37°C, 5.9% CO2, and 99% relative humidity, the
cultures were diluted 5-fold in the same medium lacking Con A. Cultures
were subsequently rediluted as needed to maintain a density of 0.3–2 ⫻ 106
cells/ml. Splenocyte cultures were maintained for up to 14 days, and cells
were used for experimentation between days 6 and 14. Flow cytometric
analysis of cultures at days 6 and 8 demonstrated that the cells present were
virtually all T lymphocytes.
Cytoskeletal manipulations
Suspended PBT were polarized by stimulation with 100 ng/ml recombinant
human stromal cell-derived factor-1␣ (SDF-1␣; PeproTech, Rocky Hill,
NJ) for 3 min or by 2-h exposure to 100 ␮g/ml colchicine or 10 ␮g/ml
nocodazole (Calbiochem, La Jolla, CA) at 37°C. Microfilaments and microtubules were disrupted in spherical resting PBT by transferring cells
from 37°C to 4°C for 30 min. IFs were disrupted by exposure to 50 nM
calyculin A (Calbiochem) for 30 min at 37°C, followed by 30 min at 4°C.
Microtubules were stabilized by 1.5-h pretreatment at 37°C with 10 ␮M
0022-1767/01/$02.00
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L
ymphocytes in circulation are subject to repeated hydrodynamic and mechanical stress, and therefore need a cytoarchitecture that is sufficiently rigid to protect them
from damage. Emigration from circulation into tissue requires the
rapid conversion of lymphocyte cytoarchitecture from a semirigid
to a highly deformable state, as cells must undergo extensive shape
changes to penetrate the small potential spaces between endothelial cells (1, 2). During transendothelial migration, lymphocytes
become highly polarized, a process driven by rapid reorganization
of all three cytoskeletal filament systems. Cells rapidly develop a
leading edge distinguished by one or more filamentous actin (Factin)2-rich pseudopodia, as well as a distinctive tail-like trailing
edge, or uropod (3). As the uropod forms, both the microtubules
(4) and vimentin intermediate filament (IF) networks retract toward and become concentrated within it.
Because the mechanical properties of the cell are largely dictated by cytoskeletal architecture (5), the cytoskeletal changes occurring during lymphocyte polarization are fundamental to the increased cellular deformability needed for extravasation. Previous
reports have implicated microfilaments, actin-myosin contractility,
and microtubules in regulation of the rigidity of circulating leukocytes (4, 6 –13). In contrast, little is known about the function(s) of
the IF network in circulating lymphocytes, nor of the role of its
collapse during lymphocyte polarization and transendothelial migration. We examined the contribution of the IF cytoskeleton to the
mechanical properties of human peripheral blood T lymphocytes
(PBT). We find that vimentin IFs are the dominant cytoskeletal
The Journal of Immunology
taxol (Paclitaxel; Calbiochem), followed by 30 min at 4°C, with and without calyculin A treatment. Control cells remained at 37°C for an equal time
after addition of equal volumes of diluent. To confirm microtubule and
microfilament disruption by cold treatment, 100 ␮g/ml colchicine (Calbiochem) or 1 ␮M latrunculin B (Calbiochem) was added 1.5 h and 30 min,
respectively, before transfer of cells to 4°C. Cells remained largely spherical in suspension with all treatments, and viability was ⬎90% by trypan
blue exclusion.
Cell deformation and immunofluorescence
Results
PBT polarize in vitro in response to a variety of stimuli, including
the chemokine SDF-1␣. Chemokines are an important signal regulating leukocyte polarization and extravasation in vivo, and can
be used with in vitro model systems to study the rapid cytoskeletal
remodeling underlying lymphocyte polarization (16). Each of the
three cytoskeletal systems in resting PBT has a characteristic geometry (Fig. 1, a– c): microfilaments are pervasive in the submembranous region and enriched in peripheral processes such as microvilli (a); microtubules radiate from the microtubule organizing
center (MTOC) (b); vimentin IFs form a coarsely woven cage
throughout the cytoplasm (c). Cytoskeletal reorganization begins
within seconds after lymphocyte stimulation with SDF-1␣ (Fig. 1,
d– g). A tail-like projection, or uropod, develops within 1 min of
stimulation. Simultaneously, F-actin-rich pseudopodia become
prominent at the leading edge of the cell (d). The IF cytoskeleton
is rapidly reorganized from the cytoplasmic network into a condensed juxtanuclear aggregate, which forms near the MTOC and
occupies much of the uropod (e). Microtubules retract toward and
become concentrated within the base of the uropod, but to a lesser
degree than do the vimentin IFs ( f).
In contrast to microfilaments and microtubules, lymphocyte IFs
remain largely unstudied. There is no established function for the
vimentin IF network of circulating lymphocytes, nor for its rapid
redistribution and compaction during lymphocyte polarization. Because circulating lymphocytes exist in a mechanically stressful environment, we hypothesized that the IF network provides mechanical support to the cell. Therefore, we examined the contributions
of vimentin IFs to the rigidity of resting, spherical lymphocytes.
We adapted an assay (17) that assesses global cell deformability by
Confocal microscopy and deformability analysis
Samples were examined on a Zeiss LSM-410 laser-scanning confocal microscope using a 100⫻, 1.4 N.A. planapochromat objective and pinhole
settings providing optical sections of ⬃0.7 ␮m. While viewing fields of
CD45-labeled cells at an equatorial focal plane, individual cells without
apparent damage or contact with other cells were randomly selected. Selected cells were then magnified using a ⫻8 optical zoom factor, and the
smallest rectangular region of interest that could enclose the cell was drawn
while focused on the adherent surface. An automated macro then acquired
two perpendicular Z-scans (XZ and YZ profiles) crossing the geometric
center of the XY region of interest. Digitized cell profiles were imported to
Image Pro-Plus (Media Cybernetics, Silver Spring, MD), in which the
width and height of each profile were measured manually by single blind
analysis. Width/height division was done to control for inherent as well as
potential cytoskeletal manipulation-induced variations in cell diameter. A
deformability index for each cell was calculated as the average width/
height ratio of the XZ and YZ profile pair. Multiple experiments were
averaged, standard error calculated, and the data then normalized to the
control group within each experiment set (either 37°C or 4°C controls).
Projection images of cytoskeletal elements in deformed samples and chemokine- or colchicine-polarized cells were generated from serial z-sections
(⬃0.7-␮m section thickness) using Zeiss LSM-410 software. Samples
comparing the distribution and structure of vimentin and lamin B were
acquired using identical brightness and contrast settings.
FIGURE 1. PBT polarization and cytoskeletal reorganization in response to SDF-1␣. The characteristic distribution of actin microfilaments
(a, blue), microtubules (b, green), and vimentin IFs (c, red) in individual
resting PBT. Polarization and cytoskeletal remodeling of F-actin (d, blue),
IFs (e, red), and microtubules (f, green) following 3-min exposure to 100
ng/ml of SDF-1␣. Overlay of the three cytoskeletal labels (g) illustrates the
pronounced collapse of the IF network (red) to the uropod. All images are
projections generated from confocal serial sections of fluorescently labeled
cells. Bars ⫽ 5 ␮m.
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Following cytoskeletal manipulations, cells were fixed for 10 min by transfer to 6-ml polypropylene tubes containing an equal volume of 4% paraformaldehyde (PFA) in Dulbecco’s PBS without calcium and magnesium
at the same temperature. After washing twice in PBS, cells were aliquoted
into 24-well tissue culture plates (106/well) containing 12-mm No. 1 coverslips, precoated overnight at 37°C with 100 ␮g/ml poly(L-lysine) (PLL;
Sigma) in borate buffered saline, and washed with distilled water before
use. Each experiment was contained within a single 24-well plate. After
addition of cells, plates were centrifuged in a Sorvall RT6000 centrifuge at
⬃1700 ⫻ g for 10 min. In experiments using live cells, plates and centrifuge were prechilled to 4°C. Following centrifugation, plates were drained
and cells fixed to the PLL substratum by addition of 4% PFA for 20 min.
Chemokine- or colchicine-polarized cells were initially fixed in 3% PFA,
washed, allowed to adhere to PLL at 1 ⫻ g for 30 min, and then fixed to
the PLL with 2% PFA. Fixed plates were washed three times in PBS, and
cells were permeabilized for 5 min with 0.2% Triton X-100 in PBS and
blocked for 1 h in PBS containing 1% BSA and 5% normal donkey serum
(DS; Jackson ImmunoResearch, West Grove, PA) at room temperature.
Cells were labeled for 1 h at room temperature in PBS/BSA/DS with
monoclonal anti-vimentin (clone V9; Sigma), anti-CD45 (mAb 9.4; provided by X. Yu, National Cancer Institute, Bethesda, MD), or anti-tubulin
(clone B512; Sigma), alone or in combination with polyclonal goat antivimentin (Sigma) or goat anti-lamin B (Santa Cruz Biotechnology, Santa
Cruz, CA). After washing four times in PBS/BSA, samples were incubated
for 1 h at room temperature with FITC or Cy5-conjugated donkey antimouse or anti-goat secondary Abs (Jackson ImmunoResearch) in PBS/
BSA/DS. F-actin was labeled by inclusion of rhodamine-conjugated phalloidin (Sigma) with secondary Abs. Coverslips were washed four times in
PBS/BSA, dried, and mounted on glass slides using 10 ␮l of a ProLong
mounting medium (Molecular Probes, Eugene, OR). A separate aliquot of
suspended cells was permeabilized and labeled with Alexa-488-conjugated
phalloidin (Molecular Probes), and relative F-actin content was quantitated
by flow cytometry. Mouse splenocytes were processed for deformability
analysis and flow cytometric analysis of F-actin, as described above. Immunofluorescent staining was performed as above, with the following exceptions: vimentin was detected using polyclonal goat anti-vimentin C-20
(Santa Cruz), and CD45 by biotinylated rat anti-CD45 mAb 30-F11
(PharMingen, San Diego, CA) and FITC-conjugated streptavidin (Jackson
ImmunoResearch).
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INTERMEDIATE FILAMENTS LIMIT LYMPHOCYTE DEFORMATION
FIGURE 2. Analysis of PBT deformability after
cytoskeletal manipulations (a). Representative confocal XZ and YZ profiles (Z-scans) of anti-CD45labeled PBT following high g-force centrifugation
onto adhesive substratum, control (37°C) compared
with sample treated with phosphatase inhibitor calyculin A (CA) at 4°C. Dashed boxes in lower panels illustrate measurement of relative width (adherent surface) vs height of cells following
deformation. b, Flow cytometric quantitation of Factin depolymerization by cold exposure compared
with 37°C controls and cold plus 1 ␮M latrunculin
B. Data represent mean FITC-phalloidin intensity
from 40,000 cells from four donors/experiments,
⫾SEM, normalized to 37°C control. c, Projection
images of deformed cells demonstrating microtubule
disruption (top row, MT) after cold exposure compared with microtubules stabilized by taxol pretreatment. Disruption of vimentin IFs by exposure to calyculin A (bottom row, IF). Bars ⫽ 5 ␮m.
Analyzing PBT rigidity using the conditions described above,
we find distinct changes in deformability following disruption of
different cytoskeletal elements. The combined disruption of both
the actin and microtubule cytoskeletons by cold treatment leads to
a small (⬃14%) but statistically significant increase in cell deformability (Fig. 3a). By comparison, subsequent disruption of
vimentin IFs by calyculin A results in a pronounced (additional
⬃38%) increase in lymphocyte deformability. Similar results were
obtained using live cells not fixed before centrifugation. The use of
fixed cells was necessary to permit comparisons between 37°C (all
cytoskeleton intact) and 4°C samples. As shown above, PBT Factin is very temperature sensitive. Fixation before centrifugation
allowed both 37°C and 4°C samples to be processed within a single multiwell plate without the risk of temperature-dependent
changes occurring during the procedure. Although fixed cells are
more rigid than live, presumably due to protein cross-linking, the
increase in deformability following IF disruption is nearly identical in the two preparations (Fig. 3b). These data show that IFs, in
the absence of microfilaments or microtubules, are sufficient to
provide significant resistance to deformation by external force. The
decreased rigidity observed following the codisruption of microfilaments and microtubules indicates that they also contribute to
the rigidity of the spherical cell, either independently or via interactions with each other or other cellular components, but to a much
lesser extent than do the IFs. To ensure that cold treatment was
sufficient to fully disrupt both microtubules and F-actin, cells were
pretreated with colchicine or the actin-depolymerizing agent latrunculin B; no increase in cell deformability compared with cold
treatment alone was observed (data not shown).
The rigidity of the nucleus may be an important determinant of
cellular deformability, particularly in cells such as lymphocytes
that have a high nuclear to cytoplasmic volume ratio (17). The
inner surface of the nuclear membrane is lined with a meshwork of
protein called the nuclear lamina, a function of which may be
mechanical support (21, 22). A major component of lymphocyte
nuclear lamina is the type V IF protein lamin B (23). Although the
lamin B nucleoskeleton can be disassembled by serine phosphorylation (24), this does not occur in lymphocytes during the calyculin A treatment that we used to disassemble vimentin (Fig. 4).
Furthermore, Western blot analysis shows that lamin B remains
insoluble following calyculin A treatment, while the cytoplasmic
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analyzing the extent of deformation during centrifugation. Suspended cells, either live or briefly fixed, were centrifuged at high
g-force onto PLL-coated coverslips. Cells are compressed against
the coverslip during centrifugation, and the highly adhesive PLL
surface preserves the contact imprint following centrifugation. After fixation to the PLL and fluorescent labeling of the plasma membrane, the extent of cell deformation was analyzed by confocal
microscopy. A representative example is shown in Fig. 2a, in
which the deformability of two PBT preparations is compared:
resting PBT at 37°C (all cytoskeleton intact) vs cells at 4°C pretreated with the phosphatase inhibitor calyculin A (all cytoskeleton
disrupted, see below). Analysis of the width vs height of cells in
the control preparation reveals that the normal cell is virtually
spherical, indicating significant resistance to deformation. In contrast, the specimen with all three filaments disrupted shows diminished height, and broadened width at the base, indicating deformation during centrifugation. For quantitative comparisons, we
calculated a deformability index as the average width/height from
paired XZ-YZ profiles of each cell; in the example shown, the
deformability index for the control cell is 0.93 compared with 1.66
for the 4°C plus calyculin A sample.
To evaluate the contributions of vimentin IFs and other cytoskeletal elements to lymphocyte rigidity using this assay, it was
necessary to establish experimental conditions that selectively disassembled subsets of cytoskeletal filaments while preserving
spherical shape and cell viability. Placing PBT at 4°C for 30 min
was highly effective for disrupting both F-actin and microtubules.
Cold exposure causes an ⬃80% loss of F-actin (Fig. 2b) as well as
a substantial disruption of the microtubule network (Fig. 2c), while
leaving the cells spherical and IFs intact. Vimentin IFs were disrupted by exposure to the protein phosphatase inhibitor calyculin
A. Because vimentin disassembly is regulated by phosphorylation
(18), treatment of cells with the calyculin A completely abolished
IF structure (Fig. 2c). Alternative methods for disrupting IFs, including acrylamide exposure (19) and microinjection of vimentindisruptive mimetic peptides (20), were ineffective, toxic, or impractical for use with spherical PBT. Pretreatment with the
microtubule-stabilizing agent taxol prevented microtubule depolymerization during cold plus calyculin A exposure. However, attempts to preserve F-actin during cold exposure using the stabilizing agent jasplakinolide were not successful, as cell morphology
and the distribution of F-actin became highly irregular.
The Journal of Immunology
6643
FIGURE 4. Calyculin A exposure disrupts cytoplasmic vimentin IFs,
but not nuclear lamin B IFs. Lamin B distribution (red, left column) vs
vimentin (green, center column) in controls, calyculin A ⫹ 4°C-treated, and
SDF-stimulated PBT. During PBT polarization, the nucleus becomes irregularly shaped. IFs are retracted to the uropod pole of the nucleus, while Lamin
B remains evenly distributed. Images are projections generated from confocal
serial sections of fluorescently labeled cells. Bar ⫽ 5 ␮m.
vimentin IFs are solubilized (not shown). During chemokine-induced polarization, the lymphocyte nucleus becomes irregularly
shaped without apparent change in lamin B nuclear envelope association (Fig. 4). This indicates that the nucleus can be deformed
without disruption of the nuclear lamina. These findings support
the conclusion that the measured increase of lymphocyte deformability induced by calyculin A is due to the disruption of the cytoplasmic vimentin cytoskeleton and is not a reflection of changes
in the properties of the nucleus.
It is possible that calyculin A treatment increases the phosphorylation of other cytoskeletal/structural components, and thus affects PBT deformability by means other than IF disruption. For
example, calyculin A is a potent inhibitor of myosin light chain
phosphatase, and therefore may alter actin-myosin contractility.
Our assay measures passive resistance to deformation, particularly
in experiments using fixed cells. As such, only intact major support
structures are likely to contribute to cell rigidity; the effects of
calyculin A on nonstructural proteins, or on previously dismantled
cytoskeletal elements such as the F-actin of cold cells, are unlikely
to be major factors. Nonetheless, the potential for non-IF-related
effects of calyculin A necessitated the use of alternative means of
achieving IF-free cells. As described above, several other methods
of IF disruption were not feasible in human PBT. Therefore, we
examined the deformability of cultured splenocytes derived from
wild-type (WT) and vimentin KO mice (15).
Freshly isolated splenocytes from normal mice expressed low
levels of vimentin protein by Western blot, and IF networks were
not clearly visible by immunofluorescence. After several days in
culture, vimentin expression is substantially up-regulated and
FIGURE 5. Polarization and cytoskeletal organization of cultured normal (WT, left column) and vimentin KO (right) mouse splenocytes.
Confocal micrographs showing the distribution of microtubles (red, MT),
vimentin IF (green, IF), and actin microfilaments (blue, MF). Bar ⫽ 5 ␮m.
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FIGURE 3. Quantitative analysis of PBT deformation following selective cytoskeletal disruption. a, Increased deformability of calyculin A ⫹
4°C-treated lymphocytes (no intact cytoskeleton) compared with 4°Ctreated cells (IF intact, microfilament (MF) and microtubule (MT) disrupted) and 37°C controls (all cytoskeleton intact). Data derived from 90
cells from nine experiments/blood donors, normalized to 37°C controls, ⫾
SEM. b, Fixed cells are less deformable than live cells, but show the same
increased deformability following IF disruption. Data from 30 cells from
three experiments/donors, normalized to fixed cell 4°C controls, ⫾ SEM.
splenocytes become highly polarized, with morphology and cytoskeletal organization equivalent to chemokine-stimulated human
PBT (Fig. 5). It is noteworthy that vimentin-deficient splenocytes
exhibit normal polarization, forming a uropod near the MTOC,
thus demonstrating that vimentin IFs are not required for polarization and uropod formation. To obtain a homogenous spherical
population suitable for analysis of global deformability, cultured
WT and KO splenocytes were incubated at 4°C for 2 h. Following
cold exposure, the IF distribution of WT splenocytes reverted from
a uropod-localized aggregate to an extended cytoplasmic network,
comparable to that of resting human PBT. WT and KO cells had
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INTERMEDIATE FILAMENTS LIMIT LYMPHOCYTE DEFORMATION
Discussion
Regulated rigidity is of fundamental importance to lymphocyte
trafficking and function. Lymphocytes must be sufficiently rigid to
survive the considerable physical stresses of circulation, but must
rapidly become highly flexible to exit the vasculature and enter
tissue. A prominent feature of the cytoskeletal reorganization during lymphocyte polarization is the retraction and condensation of
vimentin IFs into a dense perinuclear aggregate at the uropod (27).
Lymphocyte polarization in vitro, whether spontaneous or in response to chemokines, invariably includes the collapse of the IF
network. In vivo, lymphocytes within specific regions of spleen,
FIGURE 6. Splenocytes from vimentin KO mouse are significantly
more deformable than splenocytes from normal mice (p ⬍ 0.01). Quantitative analysis of relative deformability of spherical cultured splenocytes
normalized to WT samples. Data are from 95 cells in five independent
experiments, using splenocyte cultures derived from three pairs of normal
and KO mice, ⫾SEM.
FIGURE 7. Microtubule disruption-induced PBT polarization and IF
collapse. Projection images showing PBT polarization and IF collapse following microtubule disruption by 2-h colchicine treatment (100 ␮g/ml) at
37°C. F-actin (a, blue), microtubules (b, green), vimentin IF (C, red), and
overlay (d). Bar ⫽ 5 ␮m.
lymph node, and thymus have been reported to have collapsed IF
networks (29). These observations suggest that the redistribution
of the IFs is a fundamental feature of lymphocyte polarization, and
thus to the process of lymphocyte extravasation. However, little
had been known about the function(s) of the IF network in circulating lymphocytes, nor of the role of its collapse during lymphocyte polarization and transendothelial migration.
To determine how IF collapse during lymphocyte polarization
might relate to changes of cellular deformability, we examined the
contribution of vimentin filaments to the rigidity of spherical cells.
Our findings demonstrate that the IFs of spherical human PBT, in
the absence of microtubules and microfilaments, are sufficient to
maintain the cells’ resistance to extensive deformation. Consequently, to achieve the extreme level of deformation necessary for
transendothelial migration, the IF cytoskeleton of the circulating
lymphocyte would require modification. Because disassembly of
vimentin filaments into vimentin monomers would require significant energy expenditure, the compaction of the IF network
FIGURE 8. Microtubules do not provide resistance to deformation in
the absence of other cytoskeletal elements in human PBT. Quantitative
deformability analysis of cells with taxol-stabilized microtubules. PBT
with taxol-stabilized microtubules are as deformable as cells with no intact
cytoskeleton. Data from four experiments, four donors, 40 cells, ⫾SEM
normalized to 4°C control.
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equivalent levels of F-actin and extensively depolymerized microtubules following 4°C exposure. Splenocyte F-actin was significantly more resistant to cold-induced depolymerization than that of
human PBT, showing an average F-actin decrease of only 30%
after 2 h at 4°C. Live spherical splenocytes were subjected to centrifugation, and the relative deformability of normal and vimentindeficient cells was analyzed. The results (Fig. 6) show that vimentin-deficient splenocytes are considerably more deformable than
their normal counterparts. These data corroborate our findings in
human PBT following calyculin A disruption of IFs, and support
our conclusion that vimentin IFs contribute significantly to the
rigidity of spherical lymphocytes.
It has been suggested that the retraction of microtubules into the
uropod during lymphocyte polarization increases cell deformability and may thus facilitate extravasation (4). This conclusion is
based in part on findings that microtubule disruption decreases
leukocyte stiffness (6, 9, 25) and increases lymphocyte penetration
into confined three-dimensional matrices (4). However, due to secondary effects of microtubule-disrupting agents on leukocyte actin
microfilaments, cell motility (26), and IFs (27), it is unclear
whether microtubules directly contribute to the rigidity of spherical lymphocytes. PBT exposed to microtubule-disrupting agents
such as colchicine and nocodazole at 37°C become polarized (28)
and exhibit IF collapse comparable with that occurring during chemokine-induced polarization (Fig. 7). Therefore, we used the microtubule-stabilizing drug taxol to directly test the contribution of
microtubules to lymphocyte rigidity. Taxol stabilization of microtubules has no statistically significant effect on PBT deformability.
Cells containing only microtubule cytoskeletons deform to the
same extent as cells with no intact cytoskeleton (Fig. 8). These
results demonstrate that microtubules do not provide global structural support to the spherical lymphocyte in the absence of other
cytoskeletal elements. Thus, in contrast to IFs, the retraction of
microtubules to the uropod during lymphocyte polarization is unlikely to directly affect cell deformability.
The Journal of Immunology
crofilaments and microtubules (30). The interactions of IFs with
other cellular components and their distribution in the cytoplasm
may also contribute to their role as structural supports (20, 36). IFs
interact with the plasma membrane, the nucleus, and other cytoskeletal elements (37), and these interactions may regulate the
position of the nucleus and mediate the transmission of external
forces throughout the cell (5, 20, 36).
Our studies demonstrate that vimentin IFs form a cage in circulating spherical lymphocytes that provides their primary protection against deformation; although durable in circulation, the lymphocyte vimentin cage is rapidly collapsed during polarization/
transmigration. The collapse of the vimentin network during
chemokine-induced lymphocyte polarization is one of the most
rapid known examples of global IF reorganization. Understanding
the biochemical events underlying IF remodeling and the functions
of vimentin in other aspects of lymphocyte biology will be important areas for future study.
Acknowledgments
We thank Kenneth Yamada for helpful discussions, Phil Lucas for assistance with splenocyte cultures, and Tilmann Brotz for use of the Experimental Immunology Branch Microscopy and Digital Imaging Facility.
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without disassembly during polarization would be an efficient
and rapid means of increasing cell deformability. We thus
propose that the collapse of the IF network during lymphocyte
polarization satisfies the requirement for increased cell deformability during extravasation.
Using the same techniques, we have demonstrated that cultured
splenocytes from vimentin KO mice are significantly more deformable than their WT counterparts. These data support our findings using calyculin A to disrupt the IF network of human PBT.
Vimentin KO mice are viable and superficially normal (15); as in
other KOs, compensatory mechanisms may exist to replace some
of the normal functions of vimentin. As yet there are no reported
abnormalities in the development or function of the immune system of the vimentin KO mouse. The low expression of vimentin in
mouse splenocytes before culture, as well as the relative cold resistance of their F-actin, indicates that fundamental differences exist between mouse splenocytes and human PBT. The present studies do not address the physiologic functions of mouse leukocyte
vimentin; cultured splenocytes provided us the means by which to
compare the deformability of vimentin-rich and vimentin-deficient
lymphocytes without pharmacologic manipulations. We are currently investigating whether mouse PBT are more similar than
splenocytes to human PBT, and whether the loss of vimentin has
any physiologic consequence to mouse leukocytes, such as
changes in survival and/or tissue distribution.
The contribution of IFs demonstrated in these studies does not
dismiss the potential importance of microfilaments, actin-myosin
contractility, or microtubules to the overall mechanical properties
of human lymphocytes. Indeed, the cytoskeleton of lymphocytes
and other cells most likely behaves as a composite material (30),
in which the combined characteristics and interactions of microfilaments, microtubules, and IFs determine the mechanical properties of the cell as a whole (5, 30). The limited flexibility of the
IF cytoskeleton in circulating lymphocytes would be expected to
allow them to endure extreme hydrodynamic and mechanical
stress while still permitting sufficient cellular pliability to traverse
restricted passages, such as pulmonary capillaries, without its reorganization. Within the vasculature, reorganization of the more
labile actin cytoskeleton might rapidly change cortical stiffness,
while the vimentin IFs continue to maintain a level of core resistance to deformation. In addition to global resistance to deformation, local membrane stiffness is likely to be important for maintaining membrane integrity and distributing locally applied force
to the coarsely woven vimentin cage. Based on the location and
properties of the actin cortical cytoskeleton, it is likely to play a
major role in this function. The shallow angle (i.e., high radius of
curvature) of the cell membrane, where the cell meets the substratum in Fig. 2a, suggests that local stiffness is minimal when the
three cytoskeletal systems are disabled.
Our findings expand an emerging body of evidence that IFs
contribute to cellular mechanical stability. Fibroblasts derived
from vimentin KO mice are more fragile than normal cells (31).
Similarly, human and mouse epidermal cells with mutations in the
IF protein keratin tend to rupture when mechanically stressed (32–
34). Vimentin IFs are highly resistant to extraction and disassembly under a variety of conditions, suggesting that they are more
stable than microtubules or microfilaments within the ionic conditions of the cytoplasm. However, they are not static elements, as
their structure can be modulated by phosphorylation (18) and they
continuously undergo subunit exchange (35). The mechanical stability provided by IFs may be attributed in part to their unique
physiochemical properties. Isolated vimentin networks are flexible
at low strain, but rigidify in response to increasing external force,
and resist breakage at stress levels beyond those that disrupt mi-
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