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doi:10.1182/blood-2002-12-3791
Prepublished online July 10, 2003;
2003 102: 3262-3269
Katarina Wolf, Regina Müller, Stefan Borgmann, Eva.-B. Bröcker and Peter Friedl
and other proteases
through fibrillar collagen is independent of matrix remodeling by MMPs
Amoeboid shape change and contact guidance: T-lymphocyte crawling
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IMMUNOBIOLOGY
Amoeboid shape change and contact guidance: T-lymphocyte crawling through
fibrillar collagen is independent of matrix remodeling by MMPs and other proteases
Katarina Wolf, Regina Mu¨ller, Stefan Borgmann, Eva.-B. Bro¨cker, and Peter Friedl
The passage of leukocytes through base-
ment membranes involves proteolytic
degradation of extracellular matrix (ECM)
components executed by focalized prote-
olysis. We have investigated whether the
migration of leukocytes through 3-dimen-
sional collagenous tissue scaffolds re-
quires similar ECM breakdown. Human T
blasts and SupT1 lymphoma cells ex-
pressed mRNAof MMP-9, MT1-MMP, MT4-
MMP, cathepsin L, uPA, and uPAR as well
as ADAM-9, -10, -11, -15, and -17. Upon
long-term migration within 3-dimensional
collagen matrices, however, no in situ
collagenolysis was obtained by sensitive
fluoresceinisothiocyanate–collagen frag-
mentation analysis and confocal fluores-
cence/backscatter microscopy. Consis-
tent with nonproteolytic migration, T-cell
crawling and path generation were not
impaired by protease inhibitor cocktail
targeting MMPs, serine proteases, cys-
teine proteases, and cathepsins. Dy-
namic imaging of cell-ECM interactions
showed T-cell migration as an amoeba-
like process driven by adaptive morphol-
ogy, crawling along collagen fibrils (con-
tact guidance) and squeezing through
pre-existing matrix gaps by vigorous
shape change.The conceptof nonproteo-
lytic amoeboid migration was confirmed
for multicomponentcollagen latticescon-
taining hyaluronan and chondroitin sul-
fate and for other migrating leukocytes
including CD8
ⴙ
T blasts, monocyte-de-
rived dendritic cells, and U937 monocytic
cells. Together, amoeboid shape change
and contact guidance provide constitu-
tive protease-independent mechanisms for
leukocyte trafficking through interstitial tis-
sues that are insensitive toward pharma-
cologic protease inhibitors. (Blood. 2003;
102:3262-3269)
© 2003 by TheAmerican Society of Hematology
Introduction
The migration and recirculation of T lymphocytes through the
tissues is a multistep process that requires cell adhesion coupled
with cellular strategies to overcome physical tissue barriers.
1
The
principles of T-cell migration follow the paradigm of “amoeboid”
movement established for the single cell state of the lower eucaryote
Dictyostelium discoideum.
2,3
Amoeboid movement is a fast low-
affinity migration type driven by a roundish yet flexible cell
morphology, dynamic and polarized pseudopod protrusions and
retractions, which are independent of focal contact formation and
stress fibers.
2
Similar to Dictyostelium, migrating T lymphocytes
show an elliptoid polarized shape with a smooth yet dynamically
ruffling leading edge, and an additional trailing uropod.
4
This shape
generates fast (5-25 m/min) low-affinity gliding across surfaces
and through 3-dimensional (3D) collagenous scaffolds
4
driven by a
diffuse cortical actin cytoskeleton along the inner leaflet of the
plasma membrane devoid of focal contacts at substrate interactions
and stress fibers.
5,6
Amoeboid T-cell migration within extracellular
matrix (ECM) occurs independently of 1 integrin–mediated
adhesion both in vitro
6
and in vivo,
7
suggesting differences of the
molecular basis between the movement of lymphocytes and other
mobile cells, such as fibroblasts and tumor cells.
8,9
After transendothelial migration, T cells and other leukocytes
enter the fibrillar and reticular networks of the extracellular matrix
in peripheral and lymphatic tissues, predominantly composed of
fibrillar type I and III collagens.
10,11
In many cell types, adhesion
and cytoskeletal dynamics in collagenous tissues are coordinated
with proteolytic strategies to lower the physical matrix resistance.
Fibroblasts and tumor cells, as well as neutrophils, migrate across
ECM proteins such as gelatin and fibronectin while simultaneously
generating localized tracks of proteolytic substratedegradation,
12-15
prompting concepts on pericellular proteolysisas a cellular strategy
to overcome matrix barriers.
16,17
Pericellular matrix degradation is
mediated by secreted as well as cell-bound matrix proteases,
including matrix metalloproteinases (MMPs), serine proteases, and
cathepsins.
13,14,18
In tumor cells interacting with ECM, cell surface
MMPs and serine proteases become enriched at the surface of
leading pseudopods (“invadopodia”) to cooperate with integrins
and other adhesion receptors in a focalized manner.
12,17,18
Focaliza-
tion of ECM cleavage can occur by 2 principal mechanisms:
membrane-bound proteases such as MT-MMPs coclustering with
1or3 integrins at contacts to substrate,
18,19
or heterophilic
binding of soluble MMPs to cell surface receptors, such as MMP-2
binding to MT1-MMP or urokinase-type plasminogen activator
(uPA) to its receptor uPAR.
20,21
Whereas focal contacts recruit
proteases to substrate interactions in fibroblasts and tumor
cells,
18,19,22
it remains unresolved how pericellular proteolysis is
spatially and temporally achieved by short-lived and molecular
diffuse interactions to ECM substrate, as developed by rapidly
moving leukocytes that generate migration velocities that exceed
those of fibroblasts and tumor cells by 20- to 40-fold.
8
From the Department of Dermatology, University of Wu¨rzburg, Germany; and
the Department of Microbiology, University of Tu¨bingen, Germany.
Submitted December 17, 2002; accepted June 19, 2003 . Prepublished online
as Blood First Edition Paper, July 10, 2003; DOI 10.1182/blood-2002-12-3791.
Supported by the Deutsche Forschungsgemeinschaft (FR 5511/2-2 and 2-3).
K.W. was additionally supported by the foundation Evangelisches Studienwerk
e.V., Haus Villigst.
The online version of the article contains a data supplement.
Reprints: Peter Friedl, Department of Dermatology, University of Wu¨rzburg,
Josef-Schneider-Str 2, 97080 Wu¨rzburg, Germany; e-mail: peter.fr@mail.uni-
wuerzburg.de.
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’in accordance with 18 U.S.C. section 1734.
© 2003 by TheAmerican Society of Hematology
3262 BLOOD, 1 NOVEMBER 2003
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In monocytes, dendritic cells, and neutrophils, proteases from
different classes are constitutively expressed, including MMPs,
sheddases (eg, a disintegrin and a metalloproteinase [ADAMs]),
cysteine, and aspartatic proteases (eg, cathepsins), as well as serine
proteases (eg, cathepsin G, urokinase-type plasminogen activator,
human leukocyte elastase [HLE]).
17,23,24
Resting peripheral T
lymphocytes express a more restricted spectrum of proteases and
only upon activation do T cells up-regulate or de novo express
MMP-2, MMP-9, cathepsins, members of the PA/plasmin-system,
and HLE.
25-27
The penetration of T cells and other leukocytes
through basement membrane equivalents (matrigel) in vitro is
facilitated by active MMP-2 and MMP-9,
26,28
however it remains
subject to debate in which tissue compartments and under which
conditions matrix proteases contribute to transendothelial migra-
tion in vitro and in vivo.
29-32
Although focalized proteolysis, as established for stromal cells,
is an important mechanism supporting cellmigration through tissue
scaffolds, alternative pathways for bypassing ECM barriers might
exist, depending on cell type and ECM environment. In the present
study, we used rapidly moving T cells and other leukocytes to study
the expression of MMPsand additional proteases and their function
in migration through 3D fibrillar collagen matrices. Although we
initially aimed at the mechanisms of focalized proteolysis, we
followed up on the unexpected finding of undiminished leukocyte
migration in the presence of protease inhibitors and show how
amoeboid migration provides a nonproteolytic mechanism to over-
come 3D collagenous tissue barriers.
Materials and methods
Antibodies, cells, and cell culture
For flow cytometry, rabbit IgG-polyclonal anti–MT1-MMPAb (Chemicon,
Hofheim, Germany) and isotypic rabbit IgG (Sigma, Taufenkirchen,
Germany) were used.
Human peripheral blood mononuclear cells (PBMCs) were obtained
from peripheral blood from different healthy donors (by density centrifuga-
tion on a 1.077 g/mL ficoll gradient (Lymphoprep; Axis-Shield, Oslo,
Norway), washed, and stimulated for 3 days with 1.25 g/mLConcavalinA
(Oncogene, Schwalbach, Germany) and 100 U/mL IL-2 (Strathman Bio-
tech, Hamburg, Germany) in RPMI medium. CD4
⫹
and CD8
⫹
T cells from
stimulated PBMC cultures were obtained by positive immunomagnetic
selection.
33,34
Purity of isolated CD4
⫹
and CD8
⫹
populations was 95%-
99%, as determined by flow cytometry.
34
Human monocyte–derived
dendritic cells (DCs) were generated from peripheral blood of different
donors by cultivation in the presence of 5% autologous serum, granulocyte-
macrophage colony-stimulating factor (GM-CSF), and interleukin-4 (IL-4)
for 10 days and terminal maturation using tumor necrosis factor ␣ (TNF␣),
IL-1, IL-6, and prostaglandin E
2
(PGE
2
) after 7 days.
33
The human lymphoma CD4
⫹
T-cell line SupT1
26
was obtained from the
Deutsche Sammlung fu¨r Mikroorganismen und Zellkulturen (DSMZ,
Braunschweig, Germany). SupT1 cells and U937 monocytic cells were
cultured as suspension in RPMI medium (PAN, Heidenheim, Germany).
HT-1080/MT1 fibrosarcoma cells overtransfected with MT1-MMP
21
were
used as collagenolytic positive controls (kindly provided by Dr A. J.
Strongin, The Burnham Institute, La Jolla, CA). If not stated otherwise, all
primary cells and cell lines were cultured in medium containing 50 U/mL
penicillin and 50 g/mL streptomycin (PAN, Verwiers, Belgium) and 10%
heat-inactivated fetal calf serum (FCS) (Biowhittaker, Verwiers, Belgium)
at 37°C in humidified 5% CO
2
atmosphere. HT-1080/MT1 cells were
cultured in DMEM (PAN) containing 0.2 mg/mLG418 (Oncogene).
Collagen matrix migration assay, cell tracking, and cell viability
3D collagen matrix cultures (collagen concentration: 1.67 mg/mL) of
spontaneously migrating cells were monitored by time-lapse videomicros-
copy.
34
The collagen (Vitrogen, Cohesion, Palo Alto, CA) was resistant to
trypsin and sensitive to degradation by MT1-MMP, confirming its native
state as shown by sodium dodecyl sulfate–polyacrylamide gel electrophore-
sis (SDS-PAGE) and silver staining (C. Overall, E. Tam, unpublished, May
2002). In some experiments, multicomponent lattices were generated by
copolymerizing dermal collagen (1.67 mg/mL), human umbilical high-
molecular-weight cord hyaluronan (1 mg/mL; Sigma) and chondroitin-4-
sulfate (20 mg/mL; Sigma), as described.
35
For all cell types spontaneous
migration was investigated except U937 cells, which were stimulated by
lysophosphatidic acid (0.5 M; Sigma).
Locomotor parameters were obtained by computer-assisted cell track-
ing and reconstruction of the xy coordinates of cell paths for a step interval
of 1 (lymphocytes; Sup T1 cells)
34
or 3 minutes (U937 cells; DCs). The
average velocity and percentage of locomoting cells in a population were
calculated from each step interval of randomly selected cells divided by the
number of cells.
34
Cell viability was routinely assessed after migration experiments. Cells
were released from the collagen lattice by collagenase digestion (Clos-
tridium histolyticum collagenase type I; Sigma), stained by propidium
iodide (Sigma), and analyzed by flow cytometry.
Reverse transcriptase–polymerase chain reaction
Total RNA from ConA-blasts and Sup-T1 cells grown in liquid culture was
isolated using the RNeasy kit (Qiagen, Hilden, Germany). One microgram
of total RNA was reversely transcribed (1st Strand cDNA Synthesis Kit;
Roche Diagnostics, Mannheim, Germany) to cDNA using AMV Reverse
Transcriptase (1000 U/mL), dNTPs (1 mM each), MgCl
2
(5 mM), random
primer p(dN)
6
(80 g/mL), gelatin (10 g/mL), RNase inhibitor (2500
U/mL), and reaction buffer (10 mM Tris [tris(hydroxymethyl)aminometh-
ane]; 50 mM KCl; pH8,3). Each 0.25 g cDNAwas amplified by PCR in a
reaction using bacterial recombinant Taq DNA polymerase (12.5 U/mL),
dNTPs (0.2 mM), MgCl
2
(1.5 mM), sense and antisense primer (0.2 M
each) (primer sequences and references can be viewed in Supplemental
Table S1 on the Blood website; see the Supplemental Materials link at the
top of the online article) and reaction buffer (10 mM Tris-HCl, pH 8.8; 50
mM KCl; 0.08% NP40; Life Technologies, Karlsruhe, Germany). Unique-
ness of primer region was confirmed using the National Center for
Biotechnology Information blastn program. PCR was performed for 30
cycles. Absence of DNA contamination was confirmed by subjecting total
cell lysates (0.25 g total RNA) to PCR in the absence of reverse
transcriptase. The resulting PCR products were analyzed by electrophoresis
in a 2% agarose gel and stained with ethidium bromide (1 g/mL).
Specificity was determined by the length of each PCR product.
Protease inhibitors
Broad-spectrum MMP inhibitor BB-2516 (marimastat) was kindly pro-
vided by British Biotech (British Biotech, Oxford, United Kingdom).
BB-2516 blocks most MMPsand to some extentADAMs, atnM or low M
ranges.
36
Further protease inhibitors were aprotinin, trans-epoxysucciny-L-
leucylamide-(4-guanidino)butane (E-64), pepstatinA(all from Sigma), and
leupeptin (Molecular Probes, Leiden, The Netherlands). Inhibitor cocktail
was used at the following concentrations:marimastat, E-64,and pepstatinA
at 20 M; aprotinin 0.7 M; leupeptin 2 M (for inhibitor specificity,
established inhibitory concentration range, and references, see Supplemen-
tal Table S2).
Detection of collagenolysis
The efficiency of MMPinhibitor marimastat was controlled by zymography
using recombinant MT1-MMP (Invitek, Berlin, Germany), MMP-2, and
MMP-9 from supernatants conditioned by HT-1080/MT1 cells. Gelatin
zymograms were obtained overnight in the absence or presence of marimastat
(0.01-1 M) in enzyme buffer and developed by Coomassie blue staining.
Collagenolysis generated by live cells while migrating through the
native 3D collagen substrate was quantified by a novel fluorescein
isothiocyanate (FITC)–release assay. Two percent FITC-collagen type I
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monomers from bovine skin (Molecular Probes) were copolymerized
with rat-tail collagen (Becton Dickinson, Heidelberg, Germany; final
concentration 1.65 mg/mL)
18
and cells under phenol red–free conditions
(3 ⫻ 10
5
cells/l00 L medium/ well). After 40 hours’ culture in the absence
or presence of protease inhibitor cocktail, solid-phase collagen including
the cells was pelleted (15 000 ⫻ g, 10 minutes, 4°C), and the supernatant
was analyzed for FITC contents by spectrofluorometry. 100% values
represent total FITC recovery after collagenase digestion of cell-free
collagen lattices. Background fluorescence was obtained for supernatants
from pelleted nondigested cell-free lattices. In contrast to lymphocytes and
monocytic cells, DCs generated high levels of unspecific FITC release that
was not sensitive to protease inhibitors nor associated with the structural
remodeling of collagen fibers. Compared to cells isolated directly after
matrix polymerization, no increased cell-bound or endocytic FITC signal
was observed after 24 hours culture in FITC-collagen lattices in untreated
or protease inhibitor–treated cells using flow cytometry.
FITC-collagen monomers were degraded by recombinant MT1-MMP,
MMP-2, as well as trypsin as shown by SDS-PAGE and silver staining (C.
Overall and E. Tam, unpublished, May 2002). Whereas FITC release
detected classical collagenase, gelatinase, and trypsinlike activity, it was
most sensitive to MMP-inhibitor BB-2516 (reduction by 80% in HT-1080
cells
18
), indicating MMPs as primary yet not sole enzyme family in
degrading the fibrillar collagen migration substrate. In contrast to detection
of collagen fragments by SDS-PAGE and silver staining, FITC-collagen
release was a more sensitive approach to detect collagen degradation in live
cell samples (K.W., unpublished observations,April 2003).
Focalized cell-associated collagen degradation in situ was visualized by
copolymerizing 5% quenched (q)FITC-collagen type I monomers from
bovine skin (Molecular Probes) with nonlabeled collagen. As assessed by
confocal microscopy, the fluorescence intensity of qFITC in polymerized
collagen fibers strongly increased upon focalized proteolysis generated by
proteolytic HT-1080 cells, which colocalized with the appearance of
collagen cleavage-site specific neo-epitope mAb COL2 3/4 C (K.W. and
P.F., unpublished,April 2003).
Confocal laser-scanning microscopy
3D confocal backscatter microscopy of fixed samples was carried out on a
Leica TCS 4D system (Leica, Bensheim, Germany).
37
Dynamic sequences
from viable samples were obtained on the SP2 system (Leica) using a
temperature-controlled stage (37°C). Cells within the collagen lattice were
labeled with 1 M calcein-AM (Molecular Probes) and scanned at
20-second time intervals as 3D stacks of 4 to 8 z-sections. For dynamic
reconstruction, fluorescence, reflection and transmission signal were col-
lected simultaneously. Movies were obtained from 3-dimensionally recon-
structed image stacks over time. For digital image analysis, the National
Institutes of Health image program (software version 1.62) was used.
Results
In 3D collagen matrices, ConA-activated T cells spontaneously
developed an amoeboid migration type characterized by flexible
elliptoid morphology. The rapid and frequent shape change and
oscillatory stop-and-go patterns resulted in a nonlinear path
structure (Figure 1). We have investigated which matrix-degrading
enzymes are expressed by T cells and how these proteases
contribute to migration and associated remodeling of the collagen
fiber network.
Protease mRNAand surface expression
Primary human CD4
⫹
ConA-T blasts and, for confirmation, SupT1
lymphoma cells
26
were investigated for mRNA expression from
proteases of different classes, including MMPs, serine proteases,
and cathepsins (Figure 2 and Supplemental Table S1). Activated
CD4
⫹
T cells expressed MMP-9, MT1-MMP, MT4-MMP,
ADAM-9, -15, and -17, cathepsin L, urokinase-type plasminogen
activator (uPA) and its receptor uPAR as well as endogenous
protease inhibitors TIMP-2 and PAI-1 (Figure 2A). SupT1 cells
expressed MT1-MMP, ADAM-9, -10, -11, -17, uPA, and TIMP-2
(Figure 2A). MT1-MMP and cathepsin L were the only collag-
enases expressed by either cell type. ConA-stimulated CD4
⫹
T
blasts showed minor MT1-MMP surface expression (Figure 2B),
while MT1-MMP surface levels were identical to the isotypic
antibody in resting cells (not shown). As positive control, signifi-
cant MT1-MMP surface staining was detected on HT-1080/MT1
cells (Figure 2B). However, even at low levels of surface protease
expression, T cells might be able to focalize protease activity to
Figure 1. Spontaneous locomotion of an activated CD4
ⴙ
T cell within 3D
collagen lattice. Changes in morphology and oscillatory path development. CD4
⫹
ConA blast migrating in a 3D collagen matrix in the absence of a chemotactic
gradient. The average velocity was 6 m/min. Time is indicated in hh:mm:ss (11
minutes total).
Figure 2. mRNA and cell surface expression of proteases and endogenous
protease inhibitors in CD4
ⴙ
T cells and SupT1 cells. (A) Total RNA from CD4
⫹
T
cells stimulated with ConA for 3 days and SupT1 lymphoma cells was subjected to
RT-PCR (30 cycles). Fragment lengths conformed to the expected size. No mRNA
was detected for MMP-8, -13, cathepsin B and K in either cell type (not shown).
Positive controls were performed from HT-1080/MT1 or HT-1080/neo cells (MMP-7
and -9). RTindicatesreverse transcriptase reaction. (B) FlowcytometryofMT1-MMP
surface expression (gray histogram) in ConA-T blasts and positive control cells
(HT-1080/MT1) as compared with isotypecontrol (white histogram).
3264 WOLF et al BLOOD, 1 NOVEMBER 2003
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substrate contacts and thereby generate local collagenolysis for
tissue penetration.
Lack of in situ collagenolysis
Initial dose-response studies on broad-spectrum MMP-inhibitor
BB-2516 (marimastat) showed complete inhibition of gelatin
degradation by MMP-2, MMP-9, and MT1-MMP at low M
concentration (Figure 3A). However, the topographic arrangement
and focalization of proteolysis executed by live cells within the
collagen substrate are not appropriately detected by zymography.
Therefore, in situ collagen degradation caused by T cells in the
process of migration was quantified as the FITC release from
FITC-labeled fibrillar collagen lattices. After 40 hours of culture,
ConA-stimulated CD4
⫹
T blasts did not cause FITC release into
the supernatant above background levels. In contrast, collageno-
lytic HT-1080/MT1 fibrosarcoma cells released high levels of
soluble FITC into the supernatant (Figure 3B). For inhibition of
proteases, a broad-spectrum inhibitor cocktail was used to simulta-
neously target MMPs, MT-MMPs,ADAMs, cathepsins, serine, and
cysteine proteases.
18
Near-total abrogation of collagenolysis by
inhibitor cocktail was confirmed for HT-1080/MT1 cells as positive
control, while no effect was obtained for CD4
⫹
cells (Figure 3B).
The lack of FITC release was not caused by a decrease in T-cell
viability, as detected by flow cytometry after cell isolation by
collagenase digestion after 40 hours (Figure 3C).
To exclude minor local collagen fiber cleavage exerted by T
cells that might escape detection by FITC release, collagen lattices
containing quenched FITC-labeled collagen monomers were used
for confocal fluorescence and backscatter microscopy. At physical
contacts of polarized CD4
⫹
blasts to collagen fibers containing
quenched FITC, no focal in situ fluorescence above background
fluorescence was detected (Figure 3D, arrowheads), while signifi-
cant focal fluorescence was developed by HT-1080/MT1 cells at
interactions to these fibers (Figure 3E, arrowheads). Thus, MT1-
MMP and other proteases, although expressed by CD4
⫹
, did not
degrade the collagenous migration substrate.
Persisting migration in the presence of protease inhibitors
The contribution of enzymatic protease activity to T-cell migration
in 3D collagen lattices was investigated in the absence or presence
of protease inhibitor cocktail (Figure 4). Addition of inhibitor
cocktail neither changed the amoeboid morphodynamics of CD4
⫹
T cells (Supplemental Video 1) nor the oscillatory structure of the
cell paths (Figure 4A). Also, the steady-state migration velocity
was approximately 6 m/min for both nontreated and inhibitor
cocktail-exposed cells (Figure 4B, left). The number of migrating
cells including their stop-and-go-pattern remained unchanged
(Figure 4B, right). Likewise, T-cell migration within multicompo-
nent matrices containing collagen, hyaluronan, and the cross-linker
Figure 3. Lack of in situ collagen degradation by T blasts. (A)
Dose-dependent inhibition of MMP-2, MMP-9, and recombinant
MT1-MMP activity by BB-2615 (marimastat) in gelatin zymogra-
phy. (B) Lack of FITC release from FITC-labeled collagen matri-
ces by T cells, but not by HT-1080/MT1 cells (positive control).
ConA-stimulated CD4
⫹
T cells or HT-1080/MT1 cells were cul
-
tured in 3D collagen lattices containing 2% FITC-collagen in the
absence or presence of protease inhibitor cocktail for 40 hours.
Data show the means ⫹ SDs for 3 independent experiments.
(C) The percentage of viable CD4
⫹
cells remained unaffected
after 40 hours of culture in collagen. (D) CD4
⫹
T cell and (E)
HT-1080/MT1 cell incorporated in a 3D collagen lattice containing
5% quenched FITC-collagen. The false-color encoded confocal
FITC channel (color code inset: fluorescence channel for lowest
(⫺) and highest (⫹) intensity, respectively) of the 3D recon-
structed in focus sections (left) was superimposedontheshapeof
the cell body (right; red false color)obtainedfromthetransmission
image. In CD4
⫹
T cell, physical contact with collagen fibers
(arrowheads) did not result in increased cleavage-related fluores-
cence (blue false color; arrowheads), whereas HT-1080/MT1 cell
(positive control) generated focal FITC-fluorescence at fiber
binding sites (yellow false color; arrowheads). Fiber cleavage by
HT-1080 cells in situ was confirmed bystaining with cleavage-site
specific antibody (not shown). Imagesare representativefor 10 to
15 cells. Bars, 5 m.Black arrows, directionof migration.
Figure 4. Persisting migration of CD4
ⴙ
blasts in the presence of protease
inhibitors. Migration in 3D collagen (A-B) and multicomponent matrices (C) in the
absence or presence of protease inhibitor cocktail. (A) Digitized paths of 40 cells at
orthotopic position (2-hour tracking period; 1 representative of 3 independent
experiments). (B-C) Steady-state velocity and percentage of migrating CD4
⫹
T cells
in collagen (B) and collagen copolymerized with hyaluronan and chondroitin sulfate
(C). Data show the means of time-dependent population parameters ⫾ SDs for 3
independent experiments (120 cells).
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chondroitin sulfate did not depend on the function of matrix-
degrading enzymes (Figure 4C). Thus, activated CD4
⫹
T cells do
not engage the enzymatic activity of expressed collagenases for
their migration through 3D fibrillar collagen matrices.
Biophysics of nonproteolytic amoeboid T-cell migration
Because T-cell migration was neither accompanied by proteolytic
collagen fiber degradation or sensitive to protease inhibitor cock-
tail, T-cell trafficking through fibrillar collagen should occur
through a nonproteolytic mechanism. The biophysics of T-cell
migration within the collagen network were reconstructed at high
resolution by 4D confocal backscatter and fluorescence microscopy
(Figure 5). In both, control and inhibitor cocktail–treated cultures,
migrating Tcells exhibitedperiods of alignment along matrix fibers
(Figure 5A-B, black arrowheads; Supplemental Video 2). Propul-
sive squeezing through regions of narrow space prompted the
formation of a narrow zone of the cell body, termed constriction
ring
38
(Figure 5Av-vi; Bii; white arrowheads). Whereas forward
movement resulted from alignment of the cell body in parallel to
more linear fiber strands (Figure 5C, black arrowheads), directional
changes were frequent at regions of narrow space, forcing the cell
to circummigrate rather than degrade the obstacle (Supplemental
Video 3). Such migratory turns were caused by cell deflection
along adjacent collagen fibers (Figure 5D, arrowheads) and re-
sulted in angle changes along the length axis between leading edge
and trailing uropod (Figure 5D, inset; cyan arrow).As was apparent
from the movies that any change in cell shape, including cell
extension and fiber pushing, cell contraction and fiber pulling, as
well as outward dislocation of constraining fibers upon cell
squeezing, resulted in temporary deformation of the collagen
network structure (dislocation up to 2 m; movies 2-3). These
contact-dependent and temporary changes did not, however, gener-
ate long-lasting structural changes in matrix structure after the cell
had detached.
To analyze the dynamic nature of cellular interactions with
collagen fibers quantitatively, the cell boundary of a migrating T
cell (depicted in Figure 1) was obtained as 2D outline every 24
seconds (Figure 6A, dark blue) and superimposed onto the 3D
reconstruction of the transmigrated collagen matrix (Figure 6A,
green). Colocalization of outline and collagen fibers resulted in
bright pixels (Figure 6A, cyan; arrowheads) at different position
along the migration track (Figure 6A, red solid line). The position
of collagen fibers that were touched upon migration shows that the
volume of the cell follows precisely the predefined collagen
scaffold (Figure 6B, white fiber zones). The location and frequency
of cell-fiber guidance along the extracted cell outlines and their
regions in parallel contact with fibers (Figure 6C, black segments)
were quantified along 4 virtual tracks placed into the cell shape
from step to step in order to represent the geometry of both
maximum cell width (Figure 6D, a and d) and more narrow regions
near the uropod (Figure 6D, b and c). Along the migration path,
each of these tracks showed alternating contacts with collagen
fibers ranging from 1 to 10 m in length (Figure 6E, top).Although
each interaction segment was of short-lived nature, together they
generated a multidimensional physical scaffold for cell alignment
to at least one, frequently 2 or more, fibers simultaneously (Figure
6E, bottom). By means of elongation and, presumably, by volume
(which was not considered in this analysis yet apparent from the
movies), polarized T cells simultaneously aligned at several
positions to different fibers resulting in near-always guiding contact
by outside cues (see also movies 1-3). Because such contact
guidance determined both forward movement as well as migratory
turns (Figure 6C, asterisks), no bundling, structural degradation, or
remodeling of matrix fibers were required for fast T-cell crawling
within collagenous scaffolds. These findings contrast with the
migration-associated proteolytic degradation andremodeling of the
same collagen substrateby fibroblasts and tumor cells (Friedl et al,
8
Wolf et al,
18
Maaser et al,
35
and movies therein).
Nonproteolytic amoeboid migration in other leukocytes
Similar to T cells, a spectrum of matrix-degrading proteases is
expressed in other leukocytes, such as SupT1 cells
26
(Figure 2B),
DCs,
23
and monocytic cells.
24
Similar to T cells, however, only
background release of FITC was obtained from FITC-collagen for
these cells upon migration over an extended time period of 40
hours (Figure 7A), and no reduction of FITC release was achieved
by protease inhibitor cocktail (Figure 7A). The protease-
independence of migration in these cells was shown as an
undiminished velocity as well as number of moving cells in the
presence of protease inhibitor cocktail (Figure 7B; Supplemental
Video 4). Although the size and polarized morphology differ
between lymphocytes, monocytes, and DCs, each cell type fol-
lowed the principles of amoeboid movement, as indicated by
rapid shape change (movie 4), the formation of constriction rings
(Figure 7C and Supplemental Video 5), and concomitant cytoplas-
mic streaming through regions of narrow space (Supplemental
Video 5).
Figure 5.AmoeboidT-cell migration, physicalcell-fiberinteraction,andcontact
guidance within 3D collagen matrix in the absence or presence of protease
inhibitor cocktail. (A) Untreated and (B) protease inhibitor cocktail–teated calcein-
stained T cells. Crawling, alignment along fiber strands (black arrowheads) and
formation of constriction rings (white arrowheads). Image sequences (i-vi) represent
as a time series 8 (A) and 3 (B) minutes of observation time. (C) T-cell alignment in
parallel to matrix fibers (white arrowheads) upon forward migration (black arrow).
(D) Change in migration direction. Angle turns along the cellular length axis (cyan
arrow, small inset) and physical confinement of the cell body and uropod along
guiding collagen fibers (white arrowheads), reflecting the direction change. Bars,
5 m.
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Discussion
In contrast to fibroblasts
39
and tumor cells,
9,18
the migration of T
cells and other leukocytes within 3D collagen matrices is indepen-
dent of proteolytic ECM remodeling and therefore insensitive to
protease inhibitor treatment. Our data provide a mechanism on how
amoeboid crawling supports nondestructive leukocyte trafficking
through interstitial tissues.
8
Lack of migration-associated collagenolysis inmigrating T cells
In this study, T lymphocytes were used as prototype cells for
amoeboid movement in collagen. Although several matrix-
degrading proteases are expressed by activated CD4
⫹
T cells and
SupT1 lymphoma cells, including MT1-MMP, MT4-MMP, cathep-
sin L,ADAM-9, and -15
26,40
and although cathepsin Land MT1-MMP
are potent collagenases toward native type I collagen,
41,42
no degrada
-
tion of the fibrillar collagen substrate was caused by them upon
migration. We used a combination of physical and biochemical
approaches to detect collagenolysis in situ and inhibitor efficiency:
(1) gelatin zymography, (2) FITC-collagen release from FITC-
labeled collagen matrices, (3) focal in-situ fluorescence at T-cell
interactions to collagen fibers containing quenched FITC mol-
ecules, and (4) structural fiber remodeling using high-resolution
confocal backscatter microscopy from live cell samples. Specific
FITC release from FITC-collagen fibrils was at low levels for
CD4
⫹
and CD8
⫹
blasts, SupT1 lymphoma cells, and U937
monocytic cells and largely insensitive to the multicomponent
protease inhibitor cocktail. These values were 20- to 100-fold
lower than seen in HT-1080/MT1 fibrosarcoma cells used as
positive controls.
18,21
As exception, DCs generated significant
FITC release by mechanisms that were independent of the protease
classes targeted by our inhibitor cocktail, thereby currently preclud-
ing a meaningful use of the FITC release assay for DCs. Besides
collagenases, other enzymes generated by DCs, such as amino
hydrolases,
43
could dissolve the amid bond between FITC and
protein carrier without cleaving the collagenous backbone.
Migrating T cells neither bundled, extensively pulled, nor
reorganizedcollagen fibers nor generated focalcleavageof quenched
FITC-collagen or proteolytic matrix defects, in contrast to collagen-
olytic HT-1080 cells.
18
The protease inhibitor cocktail reduced
collagenolysis by 95%-98% and abrogated fiber remodeling and
proteolytic path generation in tumor cells.
18
In leukocytes, how
-
ever, the protease inhibitor cocktail did not alter any of the
migration parameters assessed by sensitive cell tracking, including
percentage of migrating cells, their velocity or stop-and-go-pattern,
nor their path profiles and the underlying interaction dynamics to
collagen fibers. These findings suggest that leukocytes do not need
to structurally modify the fibrillar collagen network for their
migration, thereby extending previous indirect evidence on T cells
penetrating radioactive-labeled collagen matrices
4
and neutrophils
migrating within amnionic membrane.
44
We cannot completely
exclude minor focal cleavage of collagen by biochemical means.
However, collagen backscatter imaging shows that the physical
fiber structure and position remain intact during and after T-cell
migration, whereas migrating tumor cells generate 3D matrix
defects using the same collagen substrate.
18,37
Together, these data
argue against a biophysically significant change in matrix architec-
ture by leukocytesalongtheir migrationtracks and further strengthen
concepts on differences of molecular migration strategies between
leukocytes and stromal cells.
8,9
Figure 6. Reconstruction of contact guidance of migrating T cell by collagen
fibers. (A) The outlineof the cell depictedin Figure 1was obtained every 24seconds
and superimposed on the 3D reconstructed backscatter signal of the collagen fibers.
Cell boundary (blue), 3D collagen matrix (green; 10 m in depth), and colocalization
of cell boundary and individual collagen fibers (cyan, arrowheads). The mean path
and pseudopodal extensions are shown by the solid red line. Migration occurred
towardthetop right corner (black arrow). (B) False color representation of thephysics
of the transmigrated path (white segments), calculated from pixel colocalization
between T-cell boundary and collagen fibers. (C) Extracted cell boundary (gray line)
and segments colocalized with collagen fibers (black sections). Asterisks indicate
turnsguidedbyoutsidefibercues.(D)Definitionof4virtualtracksalongthemigration
path toapproximate 4major morphological regions of a polarized Tcell; for instance,
lateral portions of the anterior head (a, d) and posterior cell parts near the uropod (b,
c). (E) Calculation of pixel series that represent black segments in (C) for each track
along the migration path (E, top graph), resulting in the cumulative number of fibers
that were simultaneously aligned along the forward moving cell edge (E, bottom
graph). These reconstruction data are representative for more than 5 independent
cells reconstructed by dynamic confocal backscatter imaging (compare movies 1-3).
Bars, 5 m.
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Amoeboid shape change: a nonproteolytic, biophysical
migration mechanism bypassing matrix barriers
Previous dynamicreconstruction on nonlabeled neutrophils,T cells,and
dendritic cells show leukocyte movement in collagen or amnionic
membrane as a contact guidance-dependent process that appears to
follow pre-existing matrix scaffolds.
4,6,44-46
We haveused improved
dynamic real-time reconstruction of fluorescently labeled cells
together with high-resolution imaging of the 3D matrix architec-
ture. The typical random paths and turns developed by T cells in 3D
collagen lattices result from stringent alignment of the cell body in
parallel to fibrils and fiber bundles that border random gaps and
trails pre-existing in the lattice. While following fibrillar guidance
tracks, the volume of the cell body reflects the preformed space of
trails of least resistance within the matrix. These reconstruction
data support previous work that suggests physical contact guidance
mechanisms to direct leukocyte migration in scaffolds indepen-
dently of proteolytic ECM breakdown.
6,44,45
In regions of narrow
space, depending on the pore size, 2 different mechanisms sustain
nonproteolytic movement. First, squeezing through narrow gaps by
adapting the cell shape and the formation of a constriction ring,
38
followed by cytoplasmic streaming through this immobile external
confinement zone. Alternatively, if matrix barriers are too dense
relative to the deformation ability of the cell (below 1-2 min
diameter; compare movie 3), a change in migration direction provide
movement around the obstacle. Hence, nonpenetrable regions are
circummigrated at near-undiminished velocity.
These hallmarks of amoeboid biomechanics are clearly retained
in migrating T cells, lymphoma cells, and monocytes. In DCs, the
ubiquitous dendrites and an even more striking morphologic
flexibility allow for sometimes grotesque deformation of the cell
body (movie 4). Although the principal elliptoid amoeboid shape
appears concealed in DCs, other amoeboid characteristics, such as
shape change in response to outside cues, cell constriction and
movement independent of proteases and 1 integrins remain
retained throughout migration.
46
Together, the data suggest that,
similar to neutrophils,
2
amoeboid features are retained in migrating
T cells, monocytes, and DCs, likewise.
Nonproteolytic leukocyte migration in vivo
Whereas direct in vivo measurements on protease function in
leukocyte migration are currently lacking, circumstantial evidence
suggests the contribution of protease-independent processes to
T-cell trafficking through the body. First, no obvious changes in
peripheral T-cell counts and redistribution are apparent in vivo during
MMP and other protease inhibitor–based therapy.
31,32,47
Second, resting
T cells are capable of entering lymphatic tissues through basement
membranes underlying blood vessels and recirculating, although they
produce little or no proteases. Third, resting as well as activated T cells
generate high migration velocities up to 30 m/min in vivo.
48
As in
3D collagen, the resulting contact times toward individual collagen
fibers in vivo are in the range of only 5 to 60 seconds (similar to in
vitro kinetics; compare Figure 6E), thereby greatly exceeding
interaction dynamics achieved by stromal cells that execute
pericellular proteolysis (contact time in the range of minutes to
hours).
8
Lastly, a lack of matrix degradation by migrating T cells is
obvious from in vivo imaging in the intact lymph node.
48
Besides leukocyte shape change and contact guidance, addi-
tional (patho)physiologic mechanisms are likely to support nonpro-
teolytic lymphocyte trafficking through 3D tissues. In vivo, regions
of loose fibrillar collagen networks support oedematous swelling
and reversible widening of matrix gaps in response to vasodilata-
tion and inflammation. Local edema is likely to generate biome-
chanically suitable pathways for rapid leukocyte trafficking inde-
pendent of proteolytic matrix degradation. In the dermis, such
loose connective tissue zones, which are closely mimicked by
collagen matrices, include perivascular spaces located along blood
vessels, adjacent to basement membranes, and in the dermal
papillae.
10,11,18
These “trafficking highways” are preferentially used
by passenger leukocytes upon acute and chronic inflammation,
such as in eczema.
10
Although the basic migration program in T
cells and other leukocytes does not require proteases for removing
matrix barriers, the next steps leading toward inflammatory connec-
tive tissue turnover and destruction remain to be investigated.
These likely require additional microenvironmentalchanges,includ-
ing the up-regulation and function of proteases in bystanding
fibroblasts, endothelial cells, and/or other leukocytes.
Implications
Amoeboid shape change and guidance along pre-existing matrix
structures provide supramolecular strategies for protease-indepen-
dent movement through different fibrillar tissue matrices. Because
rapidly moving leukocytes and, in particular, T cells establish only
short-lived substrate contacts coupled to diffusely distributed
integrins and actin cytoskeleton, it is reasonable to assume that
their particular membrane architecture may not support sufficient
receptor focalization required for contact-mediated proteolysis
seen in slower moving cell types, such as fibroblasts and tumor
Figure 7. Protease-independent migrationof other leukocytes. (A) ReleaseofFITC from FITC-containing 3Dcollagenmatrixby migrating SupT1 lymphomacells,CD8
⫹
T
blasts, U937 monocytic cells, and monocyte-derived human dendritic cells. Cells were cultured in FITC-collagen for 40 hours in the absence or presence of protease inhibitor
cocktail. Data represent the means ⫾ SD from 3 independent experiments. High nonspecific fluorescence of unclear origin was obtained for supernatants from dendritic cells
only. This fluorescence was neither sensitive to protease inhibitors nor did it reflect fiber degradation, as previously shown by confocal analysis.
46
(B) Mean velocity and
percentage of locomotingcells in theabsence or presenceof inhibitor cocktail. Data show the mean activities over time (⬎1hour) and standarddeviations from 3independent
experiments (120 cells) obtained from cell tracking. (C) Amoeboid movement of U937 cell resulting in the formation of a constriction ring (arrow) that remains unchanged in
position. Bar,10 m.
3268 WOLF et al BLOOD, 1 NOVEMBER 2003
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cells.
18
Amoeboid leukocyte movement, hence, represents a special
-
ized migration type that is resistant to pharmacotherapeutic target-
ing of MMPs and other proteases, thereby explaining why clinical
trials have not been complicated by leukocyte recirculation and
immune defects. These characteristics may provide a mechanism
for persistent tissue integrity despite abundant constitutive lympho-
cyte influx and recirculation.
Acknowledgments
We acknowledge Margit Ott for invaluable technical assistance,
Bong-Seok Song for assistance with confocal microscopy, and Jo¨rg
Geiger for expert help in fluorometric analysis. Eric Tam and Chris
Overall are acknowledged for collagen cleavage analysis.
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