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*
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
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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, 7, 8, 9, 10, 11, 12, 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 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.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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 x 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, 1x penicillin/streptomycin (all from Life Technologies), 50 µM 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 µg/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 x 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 µg/ml
colchicine or 10 µg/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 µM 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 µg/ml colchicine
(Calbiochem) or 1 µM 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
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 µg/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 x
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 x
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 anti-vimentin (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 anti-mouse 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 µl 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).
Confocal microscopy and deformability analysis
Samples were examined on a Zeiss LSM-410 laser-scanning confocal
microscope using a 100x, 1.4 N.A. planapochromat objective and pinhole
settings providing optical sections of 0.7 µm. 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 x8 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-µm 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.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. 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).
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a,
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.
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80% loss of F-actin (Fig. 2
b) 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
vimentin-disruptive 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.
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
F-actin 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).
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). Furthermore, Western blot analysis
shows that lamin B remains insoluble following calyculin A treatment,
while the cytoplasmic 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.
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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 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 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
vimentin-deficient 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.
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). 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.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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 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, 33, 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 microfilaments 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.
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Acknowledgments |
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Footnotes |
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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); SDF-1, stromal cell-derived factor-1; WT, wild type.
Received for publication June 9, 2000. Accepted for publication March 27, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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1 integrin-mediated adhesion. J. Exp. Med. 176:245. II protein kinase C as a mitotic lamin kinase. J. Biol. Chem. 269:19074.This article has been cited by other articles:
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