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Associated with Extracellular Matrix Fibronectin Provides a Stop Signal for Chemotactically Migrating T Cells1
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Department of Immunology, The Weizmann Institute of Science, Rehovot, Israel; and
Department of Internal Medicine, Meir Hospital, Kfar-Saba, Israel
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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, and two prototypic
chemoattractants, RANTES and stromal cell-derived factor-1
(SDF-1), on human CD45RO+ T cells migrating within an
ECM-like context. For this purpose, we used a newly constructed
three-dimensional gel system designed to follow, in real time, the
migration of individual leukocytes along chemotactic gradients in
vitro. We found that TNF-, which binds the ECM protein fibronectin
and lacks adhesion- and migration-promoting effects of its own, can act
as a proadhesive cytokine on T cells exposed to RANTES and SDF-1.
Furthermore, fibronectin-complexed TNF- provided anchorage signals
to the T cells as they moved directionally along chemoattractive
gradients. This effect of TNF- required an intact TNF- receptor
II subtype on the migrating T cells. The anchoring effect of TNF-
appears to be specific; IL-2, an integrin-activating proadhesive
cytokine, does not transmit stoppage signals to T cell migration
induced by RANTES. Thus, TNF- present in the ECM at sites of
inflammation may function to anchor T cells recruited to these sites by
chemotactic signals. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, TGF-ß, RANTES, and macrophage inflammatory
protein-1ß (4, 5, 6, 7). These mediators, through their
interactions with specific leukocyte receptors, may act concomitantly
with ECM-specific integrins to modulate leukocyte activation and
migration. During their migration, T cells may encounter, either
sequentially or simultaneously, a mixture of several of these
modulators (1, 8, 9). Therefore, it is important to
understand the reciprocal effects of various inflammatory agents that
may act alone, sequentially, or simultaneously on T cells migrating
within tissues.
We chose to study the effect of a major proinflammatory cytokine,
TNF-, a pleiotropic cytokine found in increased concentrations at
sites of inflammation (10), on T cell migration induced by
the chemokines RANTES and stromal cell-derived factor-1 (SDF-1).
Previously, TNF- had been shown to act as a specific proadhesive
mediator for human T cells in ECM environments (4, 11).
Hence, we postulated that TNF- may affect the navigation of T cells
migrating toward RANTES or SDF-1 depots within ECM.
Several approaches to determine physiological leukocyte migration, in
real time, on ECM-like gels in vitro have been described
(12, 13). We have recently developed a three-dimensional
(3-D) collagen (CO) gel reconstituted with fibronectin (FN) and laminin
(LN), the major adhesive glycoproteins of ECM, to follow and analyze
the dynamics of T cells migrating along localized gradients of
chemoattractive cytokines within the ECM environment (12).
Herein, we studied the effects of localized deposits of TNF- on the
chemotactic motility of T cells moving within ECM toward
chemokine depots. We found that although TNF- lacks
proadhesive and chemotactic activities on its own, ECM-associated
TNF- provides T cells with effective stop signals during their
migration along chemokine gradients created by T cell chemokines,
RANTES and SDF-1. This is an example of a nonmigratory cytokine,
which, when secreted within an ECM environment, can regulate the
chemotactic navigation of T lymphocytes toward chemokine sources.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The following reagents and chemicals were used: recombinant
human IL-2 (sp. act., 18 x 106 U/mg;
Chiron, Amsterdam, The Netherlands); recombinant human RANTES and
recombinant human TNF- (PeproTech, Rocky Hill, NJ); human
recombinant SDF-1 (R&D Systems, Minneapolis, MN); collagen type I
(CO-I) and LN (Cellagen, ICN Pharmaceuticals, Costa Mesa, CA); FN
(Chemicon, Temecula, CA); BSA (Sigma, St. Louis, MO); and HEPES buffer,
antibiotics, heat-inactivated FCS, sodium pyruvate, and RPMI 1640
(Kibbutz Beit-Haemek, Israel). Ab to human FN was obtained from
Calbiochem; rhodamine-conjugated goat anti-rabbit Abs was purchased
from The Jackson Laboratory (Bar Harbor, ME); murine mAb to human
TNF-, mAb11, was obtained from PharMingen (San Diego, CA); murine
mAb against human TNF- receptor type I and mAb against human
TNF-R II were obtained from R&D Systems; and murine mAb against
human CD45RA, IgG, was purchased from Serotec (Oxford, U.K.). PDLab-Tek
chambers (Nalgene, Nunc, Naperville, IL) were used for the T cell
migration assays.
Human T cells
T cells from human peripheral blood were isolated on a Ficoll gradient, washed, resuspended in PBS containing 3% heat-inactivated FCS, and incubated (45 min at 37°C in a 7% CO2-humidified atmosphere) on nylon-wool (Fenwall, IL) columns, as previously described (12, 14). The nonadherent cells were eluted and washed, and the remaining platelets were removed by centrifuging the cells (1200 rpm, 10 min, 18°C) in PBS containing 5 mM EDTA. To remove monocytes, we incubated the cells (2 h) on tissue culture grade petri dishes. The nonadherent cells were collected, washed, counted, and labeled with a Pan T cell isolation kit containing mAb against CD11b, CD16, CD19, CD36, and CD56. The labeled cells were then passed through separation columns (MiniMACS columns, Miltenyi Biotec). In a second round of purification, CD3+ T cells were labeled with mAb against CD45RA (Serotec), and a magnetically coupled secondary Ab was applied to metal wool S columns. Unlabeled T cells were subsequently collected and analyzed by FACS for CD45RO expression. The purified cells obtained (usually >97% CD45RO+ T cells) were cultured (3 x 106 cells/ml; 18 h, 7% CO2 humidified atmosphere) in RPMI containing 10% heat-inactivated FCS and IL-2 (200 U/ml).
T cell adhesion assays
Analysis of T cell adhesion to ECM ligands, with or without
TNF-, was determined as previously described (6).
Briefly, flat-bottom microtiter well plates were precoated with FN (0.5
µg/well), and the remaining binding sites were blocked with 1% BSA.
Next, 51Cr-labeled T cells
(105 cells in 100 µl of RPMI containing 0.1%
BSA) were added to the wells. Where indicated, PMA (25 ng/ml) was added
to the wells together with the cells. The plates were incubated (60
min) at 37°C in a 7.5% CO2 humidified
atmosphere and then gently washed. The adherent cells were lysed (1%
SDS in PBS), removed, and counted by a gamma counter (Packard, Downers
Grove, IL). To determine the adhesive role of matrix-complexed TNF-,
we added the cytokine to the wells (100 µl containing 250 pg/ml; 30
min, 37°C) that had been coated with FN. Unbound TNF- was removed
by washing before adding the cells. The results (±SD) are expressed as
the mean percentage of bound T cells from quadruplicate wells for each
experimental group.
Transwell chemotaxis assays
The migration of human T cells was examined in a 48-well
chemotaxis microchamber (Neuro-Probe, Cabin John, MD). The two
compartments of the microchambers were separated by an FN-coated (10
µg/well) polycarbonate filter (5 µm pore size; Osmonics Proteins
Products, Livermor, CA). Where indicated, TNF- (2 ng/ml) was
preincubated (1 h, 22°C) with soluble FN (10 µg/ml), and then the
solution (100 µl/well) was used to cover (60 min) the polycarbonate
filters. Alternatively, IL-2 (10 U/ml) or TNF- (250 pg/ml) was added
as soluble moieties to the upper chamber 15 min before adding the
T cells. To induce T cell chemotaxis, we added SDF-1 (150 ng/ml) or
RANTES (100 ng/ml) to the lower wells and added the
51Cr-radiolabeled T cells
(105 cells in 125 µl/well) to the upper
chambers. After completing the assay (150 min, 37°C in a humidified,
7.5% CO2 atmosphere), the filters were removed,
and the number of migrating T cells was determined by the radioactivity
recovered in the lower wells (14).
3-D gel migration matrix
T cell migration within ECM-like 3-D gels was performed and
analyzed as previously described (12). Briefly, the
migration gel medium consisted of RPMI containing CO-I, FN, and LN.
Purified CD45RO+ T cells were mixed with a drop
of the gel-like medium (drop I; 250 cells in 10 µl), and a second
drop (drop II; 8 µl) of the gel was placed 1.5 mm from the first drop
(Fig. 2
). Next, a chemoattractant depot was created by placing a third
drop of the gel-like medium (3 µl), which contained RANTES (100
ng/ml) or SDF-1 (150 ng/ml), 1.5 mm from the second drop and 3–5 mm
from the first drop. Once the three drops started to polymerize, the
drops were carefully connected (drops I and II first), using a 29-gauge
syringe needle, to form a continuous matrix. The slides were placed
inside CO2-filled chambers and incubated (35 min,
37°C) to allow the T cells to reach a steady-state dispersion in the
gel. Once the three drops started to polymerize, the drops were gently
connected to form a continuous matrix. To allow TNF- to become
complexed with FN in the gel, we preincubated the 3-D ECM gels (2 h,
22°C) with 100 pg/ml to 1 ng/ml of TNF- and the gel was applied to
drop II. These preparations are referred to here as ECM-bound TNF-.
In other experiments FN (1 mg/ml) was preincubated (2 h, 22°C) with
TNF- (1 ng/ml), and the complex was introduced into a gel consisting
of CO and LN, such that the FN ratio between the ECM components was
identical with the standard composition of the ECM-like gels. To
investigate the effect of soluble TNF- on migrating T cells, we
applied TNF- (250 pg/ml) directly to drop II immediately before
beginning the assay.
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T cell migration within these 3-D gels was monitored using an
inverted phase-contrast Diaphot Microscope (Nikon, Japan). T cell
movement within a selected focal plane of the gel proximal to the slide
was photographed with a long integration LIS-700 CCD video camera
(Applitech, Holon, Israel) and recorded on a time-lapse video recorder
(AG-6730 S-VHS, Panasonic, Japan) at 25 frames/min. T cell locomotion
was analyzed manually from played back video segments
(12). Time zero was defined as the time at which the cells
located at the edge of the test field closest to the chemoattractant
source started to spread and polarize in response to the diffusing
chemoattractant. Cellular movements were assigned as follows: randomly
migrating cells (motile cells that moved randomly in the gel or in a
direction away from the chemoattractant) and directionally moving cells
(T cells that migrated directionally at least one-third of the screen,
within 60 min after the beginning of the assay). For each interval of
the assay, T cell movement over a 10-min period was analyzed: the
phenotypes designated for 10 min included those obtained by motion
tracking between 5 and 15 min, the phenotypes at 20 min, those between
15 and 25 min, etc. The results shown were obtained from experiments in
which T cell viability was confirmed at the end of the assay to be
>95%. Where indicated, T cells were preincubated (40 min, 22°C)
with 250 µl of neutralizing mAb against TNF- receptors I and II (6
and 1.5 µg/ml, respectively). The treated T cells were then washed
and added to the migration gels. Before the migration assay, T cells
were washed and placed into the gels with or without TNF-.
Experiments were performed in a 37°C atmosphere that was maintained
throughout the entire experiment.
Analysis of T cell migration
T cell migration within the 3-D gels was videotaped upon completing the connections between the three drops and was analyzed as previously described (12). Cell images were converted to computerized movies using Adobe Premiere software (Abacus Concepts, Berkeley, CA) run on a G3 Macintosh computer (Apple Computer, Cupertino, CA). Cell locomotion was analyzed by Dynamic Image Analysis System (Soltech, Oakdale, LA). Individual cells were tracked during the entire period of the assay, and paths of individual cells were digitized and analyzed for total distance (per 1 h), speed (microns per minute), and vectorial distance traveled from their original position at time zero (15, 16). Polarization and T cell shape changes were monitored as previously described (17).
Immunofluorescent staining
TNF- was bound to the gels at 4°C and the gels were placed
on sterile glass coverslips. Next, the complexed matrix was polymerized
(45 min at 37°C), and the matrix was fixed (20 min) with 3.5%
formaldehyde in PBS. After having been washed with PBS and blocked with
2% OVA in PBS, the ECM gels were double stained (30 min, 4°C) with
1) polyclonal Ab (10 µg/ml) to human FN and rhodamine-conjugated
anti-rabbit IgG, or 2) mouse anti-human TNF- mAb and
FITC-conjugated anti-mouse IgG. Finally, we washed the coverslips,
which were then mounted onto glass slides and photographed (Kodak
TMAX-3200 film, Eastman Kodak, Rochester, NY) using a Zeiss Axiolab
microscope (Oberkochen, Germany).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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on chemokine-induced T cell adhesion
and migration
Certain proinflammatory cytokines, such as IL-2 and TNF-, can
bind to major ECM glycoproteins and can exert proadhesive effects
on T cells (2, 3, 4). Here, we studied the effects of
FN-associated TNF- on human T cell adhesion to FN triggered by
RANTES and SDF-1. Purified 51Cr-labeled
human T cells were added to wells coated with FN that was precomplexed
with TNF-. The cells were then exposed to the chemokine SDF-1 or
RANTES; the protein kinase C (PKC) agonist PMA served as a control T
cell activator. The percentage of cells that developed firm adhesion
was measured after incubation. The results indicate that TNF- by
itself, when complexed to FN, induces only marginal adhesion of T cells
to the ECM glycoprotein (Fig. 1A). In contrast, when it is
presented to T cells exposed to either RANTES or SDF-1, TNF-
substantially augments T cell adhesion to FN, compared with the
adhesion triggered by each chemokine alone. TNF- also augmented
PMA-induced T cell adhesion from 25 to 42% (adhesion to FN-complexed
TNF-). Chemokine or PMA-induced T cell adhesion to FN alone or FN
coimmobilized with TNF- was blocked by mAb
anti-4ß1 and
5ß1 integrins (not
shown). Hence, FN-complexed TNF-, which by itself has no proadhesive
effect on T cells, can exert significant adhesion-strengthening signals
to T cells exposed to proadhesive chemokines in the context of
FN.
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in the context of
matrix-bound TNF- involves diverse intracellular activation
pathways. In such environments, T cell adhesion was markedly
down-regulated in the presence of inhibitor of G protein signaling,
pertussis toxin, as well as other inhibitors of key
elements involved in integrin-mediated T cell adhesion, such as the PKC
inhibitor, GF109203X; the phosphatidylinositol-3 kinase inhibitor,
wortmannin; the tyrosine protein kinase inhibitor, genistein; the p38
mitogen-activated protein kinase inhibitor, SB203580; and PD98059, an
inhibitor of mitogen-activated protein kinase (data not shown).
The ability of TNF- to augment the chemokine-triggered adhesiveness
of CD45RO+ T cells to FN suggests that this
cytokine may also modulate the migration of these cells through
FN-containing extravascular spaces. Hence, we analyzed the effects of
soluble or FN-complexed TNF- as well as the effects of a control,
proadhesive cytokine, IL-2, on T cell migration along RANTES or
SDF-1 gradients in conventional chemotaxis chambers. The results
indicate that IL-2 does not have any effect on T cell migration toward
both chemokines. In contrast, soluble TNF- and, to a greater degree,
FN-bound TNF- significantly inhibit the RANTES- and
SDF-1-induced T cell migration (Fig. 1
B). Thus, the
adhesion-strengthening, integrin-dependent signals transmitted to T
cells by FN-interacting TNF- in the context of proadhesive
chemokines interfere with T cell chemotaxis to these chemokines.
Analysis of T cell migration induced by matrix-associated
TNF-
To examine and analyze the effects of matrix-complexed TNF- on
the migratory properties of individual CD45RO+ T
cells, we followed in real time T cell migration within chemotactic
gradients formed in 3-D ECM-like gels containing CO, LN, and FN
(12). T cell migration along chemotactic gradients of
either RANTES or SDF-1 was tracked in intact ECM gel as well as in a
gel into which TNF- had been introduced in different states. A
scheme of the setup, depicted in Fig. 2, illustrates the relative positions of the T cells, the migration zone,
and the chemokine depot before the beginning of the assay.
When TNF- was introduced into the migration zone without the
chemoattractant in the depot drop, a significant fraction of the T
cells polarized and randomly migrated short distances within the gel
(Fig. 3A). Leukocyte
polarization is an early activation-dependent event linked to cell
adhesion and motility (12, 16). Indeed, most of the T
cells embedded in the gel without any cytokine remained spherical and
nonmotile throughout the entire assay period. T cell polarization was
abrogated by the depletion of the integrin ligands FN and LN from the
cytokine-containing gels (12) (data not shown). T cell
motility in the ECM-like induced by TNF- steadily decreased over
time (Fig. 3A). This time-dependent effect of TNF- could
reflect changes in the biochemical state of TNF- within the gel at
different time periods. To resolve this question, we immunostained the
gels with FN- and TNF--specific Ab to monitor the distribution of
TNF-. Within 40–60 min, most of the TNF- became codistributed
with the FN constituent of the gel; both molecules were found to
colocalize in fibrillar and globular patterns (Fig. 3C),
typical of the structure of FN, which consists of two distinct
patterns: dense globular aggregates and thin fibrillar strands. Thus,
soluble TNF- added to the gel can rapidly associate with the FN
fibrils. The time-dependent decrease in motility observed in this
experiment suggests that T cells respond differently to de novo
secreted soluble TNF- as opposed to FN-complexed TNF-. Indeed,
when FN was precomplexed with TNF-, and the complex was then added
to the LN- and CO-containing gel, the T cells introduced into the gel
immediately polarized and remained nonmotile over the entire period of
the assay (Fig. 3B).
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In light of the profound proadhesive effects of FN-complexed
TNF- on chemokine-stimulated T cells (Fig. 1A), we
questioned whether TNF- introduced into a migratory zone of T cells
chemoattracted by specific chemokines can modulate the migration of the
cells. To this end, T cells were placed in drop I, the chemokine was
added to drop III, and T cell migration within a migration zone of
intact ECM-like gel or a gel containing the FN-TNF- complex (in drop
II) was analyzed. First, we assessed the dose-response, chemoattractive
effects of RANTES or SDF-1, present in drop III at the beginning of
the assay in the absence or the presence of TNF- in drop II. The
results, shown in Table I, indicate that
in the absence of TNF-, the effective chemoattractive concentrations
of SDF-1 and RANTES were 150 and 100 ng/ml, respectively. When
TNF-, at 0.1–1 ng/ml, was prebound in drop II, a marked inhibition
of T cell chemotaxis occurred. The amount of 250 pg/ml for TNF- was
chosen based on a previous study in which we found that 4–6% of the
soluble TNF- actually bound FN and elevated the PMA-induced T cell
adhesion (11).
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is
shown in Fig. 4. In the absence of
TNF-, RANTES induced a marked directional T cell migration oriented
along the developing gradient, within up to 60 min, followed by an
apparent decline in T cell migration. This is probably due either to
the cells reaching a field of homogeneous dispersion of chemokine
molecules within the ECM or to chemokine receptor desensitization
(18). When TNF- was present in the migration zone, an
almost complete suppression of T cell migration toward RANTES was
observed (Table I and Fig. 4). The inhibitory effect of TNF- on
migration was not due to the apoptosis-inducing capacities of the
cytokine, because the migrating T cells remained intact throughout the
studies and thereafter (not shown). To determine whether the
migration-inhibiting effect of TNF- is due solely to its
adhesion-strengthening effects on T cells interacting with FN, agonists
of ß1 integrins, such as PMA or IL-2
(14), were introduced into drop II as control activators.
Surprisingly, IL-2 did not alter the proportion of
RANTES-induced, directionally migrating T cells. PMA, a powerful PKC
activator capable of locking integrins in high-avidity states, reduced
the number of T cells migrating toward RANTES, similar to
immobilized TNF- (Fig. 4).
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or soluble
TNF-, introduced to the migration zone simultaneously with the
generation of chemokine gradient (Fig. 2), to modulate the migratory
properties of T cells moving along chemokine gradients. Consistent with
the strong inhibitory effect of TNF- complexed to FN on the
directional migration of T cells toward RANTES (Fig. 4),
matrix-associated TNF-, but not the soluble cytokine, abolished the
directional migration of T cells along RANTES or SDF-1 gradients (Fig. 5, A and B,
respectively). The chemokine- or TNF--induced T cell responses in
the 3-D gel (e.g., polarization, random and directional movements),
within the indicated time frame (10–60 min), were not the result
of alterations in the surface expression of the relevant chemokine
receptors (i.e., CCR5 and CXCR4) as well as FN-specific
ß1 integrins, as confirmed by FACS staining of
these molecules after T cell exposure to TNF- for 15–60 min (data
not shown).
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depicts representative pathways of T
cell migrating toward RANTES. Whereas no directional migration of T
cells was observed in either intact (Fig. 6A) or
TNF--containing migratory zones without a chemokine gradient (Fig. 6B), highly directional migration of T cells was observed
along RANTES gradients, with a high ratio between the vectorial
distance and the total distance traveled by an individual cell from the
origin of its migratory path (Fig. 6C). Note that T cells
located close to the RANTES depot exhibited low migration rates,
suggesting that a directional migration requires a chemoattractant
gradient rather than a high local concentration of the chemokine. T
cells migrating along the RANTES gradient in the presence of
matrix-associated TNF- were highly heterogeneous in their pathways
(Fig. 6D). A fraction of the cells migrated short distances
in the direction of the RANTES gradient before coming to a complete
stop. Another small fraction of T cells migrated long distances toward
RANTES, similar to the migratory properties of T cells migrating
toward RANTES in the absence of TNF- (Fig. 6C). A third
fraction of T cells retained their ability to migrate directionally
toward the chemokine, but their migration paths were shortened in the
presence of TNF- (Fig. 6D).
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, was performed
with T cells from three donors. Matrix-associated TNF- markedly
decreased both the total distance and the vectorial path lengths of all
tested T cells present in the migration zone (Fig. 7A). These significant effects
were TNF- specific, because the total distance traveled by the
RANTES-attracted T cells was only minimally reduced if IL-2 was present
in the migration zone, instead of TNF-. Nevertheless, IL-2 in the
migration zone significantly reduced the directionality of the
RANTES-induced T cell chemotaxis (Fig. 7B and data not
shown). The microdynamic analysis revealed that the velocity and
directionality of cells moving along RANTES gradients were not
significantly altered until being stopped by TNF- (Fig. 7B). Thus, the shorter distance traveled by cells toward
RANTES in the presence of TNF- was due to the reduction in the
duration of migration, rather than to the speed or the directionality
of migration. Indeed, the mean duration of T cells migrating toward
RANTES dropped from 65 ± 15 min in the absence of TNF- to
32 ± 4 min in the presence of TNF-. Similar findings were
observed in T cells migrating along SDF-1 gradients; TNF- reduced
the mean duration of directionally migrating cells from 45 ± 5 to
32 ± 7 min. In conclusion, the cell paths depicted in Fig. 7B indicate that the T cells move directionally along the
chemotactic gradient until encountering TNF--rich areas that rapidly
triggered their complete arrest. Thus, TNF- and IL-2 reduced the
directional distances traveled by T cells toward a chemotactic source
through distinct mechanisms: TNF- by stopping a previously migrating
cell, and IL-2 by constantly reducing the directionality of the
locomoting cell.
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are mediated by
TNF- receptor type II
TNF- is recognized by two receptors, designated TNF-R-I and
-II, which, upon ligand binding, transmit differential intracellular
signals (10, 19, 20). We examined the contributions of
both receptors to the stop signal exerted by FN-bound TNF-. T cells
were pretreated with mAb specific for TNF-R-I or -II and then
introduced into the RANTES-containing gel in the presence of
immobilized TNF-. Anti-TNF-RII but not anti-TNF-RI mAb completely
abolished the inhibitory capacity of the cytokine on both the number of
T cells migrating toward RANTES (Fig. 8)
as well as on their directionality of migration, as reflected in the
microdynamics of their motions (not shown). Hence, the
migration-stopping effect of TNF- is transmitted to the
chemotactically migrating T cells exclusively through their TNF--RII
subtype. Interestingly, both mAbs blocked the proadhesive effect of
this cytokine on chemokine-induced T cell adhesion to FN in a static T
cell adhesion assay (not shown). Thus, there is no direct relationship
between the proadhesive effects of a cytokine signal mediated by both
TNF- receptors and the unique role of one of these receptors, namely
TNF-R-II, in delivering stoppage signals to the migrating T
cells.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We initially observed that FN-complexed TNF- can suppress the
chemotactic migration of human CD45RO+ T cells
toward potent chemokines, such as SDF-1 and RANTES in the Transwell
system (Fig. 1). Having observed this suppressive effect of TNF-, we
next investigated how the chemotactic navigation of individual T cells
is affected by the presence of TNF-. We found that FN-associated
TNF-, although lacking chemotactic activity of its own (Table I and
Fig. 3), can transmit signals to T cells to stop their migration along
chemokine gradients.
The mechanism underlying the stoppage effect of TNF- on T cell
migration within ECM is not known. Such stoppage may result from
modulation of the expression or rapid activation of ECM-specific
adhesion receptors on the migrating leukocytes. However, TNF- did
not alter the expression of ß1 integrins (or
SDF-1 and RANTES receptors) on T cells during the time frame of the
assay or even during longer periods (not shown). In addition, it is
unlikely that TNF- transmits such a rapid stoppage signals to T
cells by inducing the de novo biosynthesis of a stoppage factor by the
migrating T cells. In light of its ability to strengthen the
agonist-induced ß1 integrin-mediated adhesion
of T cells to FN during this time frame, we propose that the stoppage
effect of TNF- involves modulation of ß1
integrin adhesiveness triggered by chemokines during the migratory
process. Indeed, T cell adhesion induced by both immobilized TNF-
and chemokines involved diverse intracellular signaling events, which
are all linked to the stimulation of ß1
integrin avidity and adhesion strengthening (manuscript in
preparation). Moreover, adhesion-strengthening effects induced by PMA
or integrin-activating mAb also resulted in complete stoppage of T
cells present within chemokine gradients (Fig. 4 and data not shown).
Interestingly, TNF- alone failed to strengthen
ß1 integrin-mediated T cell adhesion both in
prolonged static assays (Fig. 1) (11) and during short
static contacts, during which chemokines could stimulate cell adhesion
(R. Alon, unpublished observation). Hence, TNF- appears to
strengthen cell adhesion primed by chemokine-stimulated integrins,
thereby, generating high-avidity interactions with ECM and stoppage of
cell motility. Herein, the matrix-complexed TNF- was found to reduce
the motility of T cells and transmit stop signals to
chemokine-attracted T cells much more efficiently than the soluble form
of the cytokine (
Figs. 3–6). We propose that although the absolute
amounts of the FN-bound cytokine are diminutive, the lymphocytes could
have actually encountered high localized amounts of the cytokine, as
indeed suggested by our FN and TNF- colocalization findings (Fig. 3C).
Is the ability of TNF- to transmit a stop signal unique to this
cytokine? Proadhesive, matrix-associating cytokines were reported to
exert antimigratory effects on leukocytes migrating along chemotactic
gradients (24). TCR engagement transmitted a stop signal
to T cells migrating on ICAM-1 in vitro (23). In contrast,
TNF- differs from most other cytokines in that it does not trigger T
cell adhesion or migration on its own, but, rather, strengthens the
adhesion triggered by other agonists. The results shown in Figs. 3 and 6 indicate that TNF- can actually activate T cells in the context of
ECM, because it induced T cell chemokinesis and polarization. Yet, as
shown in our studies (Figs. 1C, 3, and 6), TNF-, in its
matrix-bound or soluble forms, lacks a detectable chemotactic effect.
Consistent with our findings (Fig. 3), it has been previously shown
that TNF- can induce the polarization, but not the locomotion, of
neutrophils (29). In contrast to TNF-, IL-2, a
proadhesive and promigratory cytokine by itself (12),
which, similar to TNF-, can interact with ECM proteins
(14) and regulate the expression of chemokine receptors on
leukocytes (30), did not induce a stoppage effect on T
cells (Fig. 4). Thus, we not only could demonstrate that the ability to
deliver stoppage signals is unique to TNF-, but could also show that
proadhesive effects and stoppage signals, delivered to ECM-interacting
T cells by cytokines, are separately regulated.
TNF- is recognized by TNF--RI and -II on various cell
types, including leukocytes (19, 31). These two receptors
differ not only in their TNF--binding capacities and functions, but
also in their expression. In contrast to the constitutive level of
expression of TNF-RI on leukocytes, the expression of TNF-RII requires
cell activation (10). Our results suggest that TNF-RII on
activated CD45RO+ T cells is the TNF--R that
exclusively mediates the cytokine’s capacity to stop T cell migration.
This may be due to the preferential binding of FN-complexed TNF-,
rather than soluble TNF-, to TNF-RII. Interestingly, TNF-RII, but
not TNF-RI, modulates the migration of epidermal Langerhans cells in
vivo (32).
Chemoattractive gradients and proinflammatory cytokines can be
continuously formed by tissue-resident, as well as newly invasive,
immunocytes (18, 32, 33, 34, 35). The migration of T cells in
tissues may be directly induced by chemoattractive cytokines or
bacterial Ags (25, 27) or indirectly induced by the
capacity of cytokines to stimulate the secretion of promigratory
mediators for lymphocytes by various cells at the site of inflammation
(36). Our study simulated an inflammatory scenario within
an ECM-like environment in which T cells were attracted by a de novo
generated chemoattractant gradient encountered within a migratory zone
into which TNF- had been previously deposited. This scenario assumes
that TNF- became associated with the ECM before the chemokine
gradient was formed. It would then function as a strong and rapid
adhesion-strengthening moiety (37, 38) and anchor the
chemoattracted T cells at these sites.
The idea that some chemoattractants may inhibit chemotactic migration induced by other attractants has been recently suggested and studied by Butcher et al. (8, 9). Pre-exposure of leukocytes to a dominant attractant, such as the peptide fMLP, strongly suppressed the ability of these cells to migrate, in a non-ECM context, toward a secondary and weaker chemoattractant, IL-8. Butcher et al. proposed that leukocytes navigate in the tissue by responding to a complex chemoattractant field in a step-by-step manner, such that their ultimate positions are determined by the sequence of chemoattractive information encountered during their migration. Our results extend this idea in that not only do different attractants control the navigation of cells to other attractants at the site of inflammation, but nonmigratory cytokines can also regulate the final localization of lymphocytes at chemotactic fields. Therefore, the type of cytokine, its amount, its context, and the order of presentation to the migrating leukocyte may determine the ultimate position of the locomoting cell within tissue. This information is translated within the responding T cell to directional migration, random movement (chemokinesis), or arrest.
Migration involves the regulated function of cytoskeletal elements and
integrins (39, 40). The relationship between proadhesive
information and motility is complex; optimal cell motility requires a
medium level of avidity between the migrating cell and adhesive
proteins in the migration matrix (15, 39). Therefore, it
is difficult to predict how a cytokine with both proadhesive and
chemokinetic properties would determine T cell motility in a given
environment. An intriguing possibility raised by our findings of the
unique stoppage signals transmitted by TNF-, but not by IL-2, is the
existence of a hierarchy between different types of nonchemotactic
cytokines with respect to their abilities to modulate T cell migration
in a chemotactic context. Future studies should be aimed at examining
the possibility that other inflammatory cytokines can also inhibit T
cell chemotaxis. In this respect it has been found that a prolonged
incubation (i.e., 4–20 h) of monocytes with bacterial Ags or IFN-
blocks their migration on a monocyte chemoattractant protein-1-formed
chemotactic gradient by selectively down-regulating the expression of
the monocyte chemoattractant protein-1 receptor CCR2 (41, 42). However, the mechanism underlying the rapid stoppage effect
of TNF- cannot be attributed to modulation of chemokine or integrin
receptor expression. It is also reasonable to assume that this mode of
rapid T cell activation by TNF- is not sufficient to affect gene
transcription or RNA translation of these receptors.
A putative role for TNF- secreted and integrated into the ECM at
sites of inflammation may be to anchor momentarily the directional
migration of T cells to these sites, where their effector functions are
required. Such an anchorage of T cells by ECM-complexed TNF- may be
regarded as a checkpoint in activation processes of the migrating
lymphocytes (23). The in vivo consequences of such
stoppage may be either proinflammatory (e.g., retention of immune
cells) or suppressive (e.g., attenuating excess recruitment of effector
cells), depending on the context of chemoattractive information
presented to the locomoting cells within the specific ECM
microenvironment.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Drs. Ofer Lider and Ronen Alon, Department of Immunology, The Weizmann Institute of Science, Rehovot 76100, Israel.
3 Abbreviations used in this paper: ECM, extracellular matrix; 3-D, three-dimensional; CO, collagen; FN, fibronectin; LN, laminin; SDF-1, stromal cell-derived factor-1; PKC, protein kinase C.
Received for publication March 23, 2000. Accepted for publication June 16, 2000.
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