| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Departments of
*
Immunology and
Organic Chemistry, Weizmann Institute of Science, Rehovot, Israel; and
Department of Hemato-Oncology, Sheba Medical Center, Tel-Hashomer Hospital, Israel
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
IL-2 is a 15.5-kDa glycoprotein that participates in the development of
inflammation and in the regulation of apoptosis (7, 8). In addition to
its proactivatory and proliferative roles, IL-2 also induces neutrophil
adhesion to HUVEC in a CD18-mediated manner (9) as well as
chemotactic responses in T cells, both directly and by regulating their
expression of CC chemokine receptors (10, 11). The IL-2R consists of
three distinct membrane chains: the 
-, ß-, and 
-chains. The
ability of IL-2 to induce T cell activation, differentiation, and
proliferation involves the ß- and -chains of the IL-2R, which are
coupled through their cytoplasmic domains to intracellular protein
tyrosine kinases and a protein serine/threonine kinase (8, 12). X-ray
crystallographic analysis and deletion experiments showed that the
sites of IL-2 that bind to the ß- or -chains of its receptor are
located within the -helical and 30-amino acid residues of the
N-terminal domain of IL-2 (13). Indeed, anti-IL-2 Abs that
recognize amino acid epitopes in the N-terminal region of IL-2 can
inhibit IL-2-induced lymphocyte proliferation. The C-terminal portion
of IL-2 and its three Cys residues seem to contribute to the folding
and active conformation of IL-2 (13, 14).
Most neutrophil elastase (also termed human leukocyte elastase), which
exists as either a membrane-bound or soluble moiety, is produced and
released by neutrophils, although small amounts are also produced by
macrophages, monocytes, and T cells (15, 16). Elastase degrades
basement membrane and ECM glycoproteins, such as elastin, collagen, and
fibronectin (FN), as well as molecules expressed on the surface of T
cells, e.g., CD4, CD8, and CD2 (17). Recently, two novel functions of
neutrophil elastase have been shown: 1) membrane-bound elastase
modulates immune cell adhesiveness by interacting with the integrin
Mß2 on neutrophils (18); and 2) elastase
processes IL-8 and thus alters the biologic functions of this chemokine
(19).
Recently, we have shown that IL-7, among other cytokines, interacts
with ECM, and that both soluble and matrix-complexed IL-7 induce
integrin-mediated adhesive interactions of human T cells with ECM-bound
IL-7 and with purified VCAM-1 molecules (20, 21). The biologic effects
of IL-7 are linked to its interaction with the -chain of the IL-2R,
which is a tyrosine kinase signal-transducing molecule (22). Also,
exposure of T cells (3 to 5 days) to IL-2 was found to induce their
migration on collagen (23). Therefore, we examined whether IL-2 can
induce T cell adhesion to ECM glycoproteins, whether elastase can
process human rIL-2, and whether such putative IL-2 peptides can affect
T cell-ECM interactions. We found that IL-2 can indeed induce T cell
adhesion to ECM glycoproteins that are otherwise nonadhesive moieties.
In addition, we may have identified a group of naturally occurring,
elastase-generated IL-2 fractions and peptides capable of inhibiting T
cell adhesion and migration.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The following reagents were obtained as indicated. Human rIL-2 (sp. act. 18 x 106 U/mg; Chiron, Amsterdam, The Netherlands); human rIL-7 (sp. act. 2 x 105 U/µg; Immunex, Seattle, WA); recombinant human MIP-1ß (PeproTech, Rocky Hill, NJ); FN (Chemicon; Temecula, CA); BSA, laminin (LN), PMA, and TRITC-conjugated phalloidin (Sigma Chemical, St. Louis, MO); collagen type IV (CO-IV; ICN, Costa Mesa, CA); and HEPES buffer, antibiotics, heat-inactivated FCS, sodium pyruvate, and RPMI 1640 (Beit-Haemek, Israel). An anti-ß1 integrin-specific affinity-modulating mAb, 8A2 (24), was donated by Dr. J. M. Harlan (Washington University, Seattle, WA). All protected amino acids, coupling reagents, and polymers were obtained from Nova Biochemicals (Läufelfingen, Switzerland). Synthesis-grade solvents were obtained from Labscan (Dublin, Ireland). HPLC solvents and columns were obtained from Merck (Darmstadt, Germany).
T cell adhesion assays
Human T cells were purified from the peripheral blood of healthy donors, and T cell adhesion to immobilized protein substrates was examined as previously described (20, 21). Briefly, human leukocytes were isolated on a Ficoll gradient, washed, and incubated (2 h, 37°C, 7.5% CO2, humidified atmosphere) on petri dishes. The nonadherent cells were then collected and incubated (1 h, 37°C, 7.5% CO2, humidified atmosphere) on nylon wool columns (Novamed, Jerusalem, Israel). Unbound cells were eluted from the columns by extensive washings. The resulting cell population was always >92% T cells. The flat-bottom microtiter wells that had been precoated with ECM or ECM proteins (FN or LN; 1 µg/well) were blocked with 0.1% BSA. After 0.5 h at 37°C, the wells were washed and 51Cr-labeled T cells were added to the wells, 105 cells/100 µl of adhesion medium (RPMI 1640 supplemented with 0.1% BSA, 1% sodium pyruvate, 1% HEPES buffer). The microtiter plates containing the cells were incubated (30 min, 37°C) in a humidified, 7.5% CO2 atmosphere and then washed. The adherent cells were lysed, and the resulting supernatants were removed and analyzed in a gamma counter. For each experimental group, the results were expressed as the mean percentage ± SD of bound T cells from quadruplicate wells. To some wells, different concentrations of soluble IL-2 were added concomitantly with the T cells, and with others, different concentrations of elastase-degraded IL-2-derived fractions, or the corresponding synthetic peptides, were added together with stimulators (PMA (50 ng/ml), IL-7 (50 ng/ml), MIP-1ß (20 ng/ml), 8A2 (1 µg/ml), mAb anti-CD3 (1 µg/ml), or IL-2 (10 U/ml)).
Chemotaxis assays
T cell chemotaxis was performed and analyzed as previously described (25). Briefly, the migration of human T cells (0.5 x 106 cells in adhesion medium/well) was examined in a 48-well chemotaxis microchamber (NeuroProbe, Cabin John, MD). The two compartments of the microchambers were separated by a FN-coated polycarbonate filter (5-µm pore size; Osmonics Protein Products, Livemore, CA). Where indicated, MIP-1ß or IL-2 was added to the lower wells, and the T cells were added to the upper chambers together with the peptides. After incubation (120 min, 37°C, in a humidified, 7.5% CO2 atmosphere), the filters were removed, fixed, and stained with a Diff-Quik staining kit (Dade, Düdingen, Switzerland). The number of migrating T cells in five high-power fields (under 500x magnification; Wild Microscope, Heerbrugg, Switzerland) was evaluated. For each group, the results are expressed as the mean number of cells in one high-power field.
Purification of elastase and elastase digestion of IL-2
Neutrophils were isolated from the whole blood of a healthy donor by dextran sedimentation and Ficoll-Hypaque gradient centrifugation, as previously described (26). Elastase was isolated by aprotinin-Sepharose affinity chromatography, followed by carboxymethyl-cellulose ion exchange chromatography as developed by Baugh and Travis (26, 27). The purified elastase, which was lyophilized and stored at -20°C until used, was biochemically checked to be entirely free from cross-contamination with cathepsin G (not shown). IL-2 was dissolved in distilled water to yield a 1 mg/ml solution. Lyophilized neutrophil elastase (50 µg) was dissolved in 1 ml of PBS and immediately added to the IL-2 solution. The elastase-IL-2 mixture was incubated (12 h) at 37°C. Aliquots were removed and stored at -20°C until subjected to HPLC separation.
Reverse phase HPLC
Elastase digests of IL-2 were purified with a prepacked Lichrospher-100 RP-18 column (4 x 25 mm, 5-µm bead size), using a binary gradient formed with 0.1% trifluoroacetic acid (TFA) in H2O (solution A) and 0.1% TFA in 75% acetonitrile in H2O (solution B; at t = 0 min, B = 3.5%; at t = 5 min, B = 3.5%; and then the concentrations began to increase: at t = 60 min, B = 100% (i.e., 75% acetonitrile)). The flow rate was constant at 0.8 ml/min. A Spectra-Physics SP8800 liquid chromatography system (Fremont, CA) equipped with an Applied Biosystems model 757 (Foster City, CA) variable wavelength absorbency detector was used. The column effluents were monitored by UV absorbency at 220 nm, and the chromatograms were recorded on a ChromeJet integrator. Fractions that were 20% or more above valley levels were pooled, rotoevaporated to a minimal volume, and diluted with HPLC grade water. The rotoevaporation and dilution with water step was performed twice to remove residual TFA and acetonitrile.
Amino acid composition of the synthetic peptides and amino acid sequence analysis
Purified peptide solutions (
40 µg of peptide in 40
µl, with 5 µg of norvaline (an unnatural amino acid) as an internal
standard) were rotoevaporated, hydrolyzed (10°C, 22 h) in 6 N
HCl under vacuum, and analyzed with an amino acid analyzer (HP1090,
Hewlett-Packard, Palo Alto, CA). An on-line precolumn
o-phthalaldehyde/9-fluorenylmethoxycarbonyl (F-moc)
derivatization, combined with reverse phase chromatography, was used to
determine the amino acid composition of the peptides and the total
peptide yield. Without exception, all of the peptides yielded excellent
analysis ratios of corresponding amino acid deviations from expected
values of <10%.
Analysis of the elastase-generated IL-2 fractions was performed using an Applied Biosystems model 470A gas phase microsequencer. Phenylthiohydantoin amino acid derivatives were separated on-line by reverse phase HPLC on a PTH C-18 column (2.1 x 220 mm) using a model 120A analyzer (Applied Biosystems).
Solid phase peptide synthesis
IL-2-derived peptides were prepared by conventional solid phase peptide synthesis, using an AMS-422 automated solid phase multiple peptide synthesizer (Abimed, Langenfeld, Germany). The F-moc strategy was used for peptide chain assembly according to the commercial protocol. In each reaction vessel, we used 12.5 µmol of Wang resin containing the first covalently bound corresponding N-F-moc C-terminal amino acid (typically, polymer loadings of 0.3–0.7 mmol/g resin were used). F-moc deprotection was achieved by two consecutive treatments with 20% piperidine in dimethyl formamide, usually 10 to 15 treatments each min at 22°C, depending on the length of peptide and the F-moc-protected amino acid type. The protecting groups used for the side chain of the amino acids were tert-butyloxycarbonyl for Trp, trityl for Asn, and, tert-butylether for Thr. Coupling was usually achieved using two successive reactions (typically 20–45 min each at 22°C, depending on the length of peptide and amino acid derivative type) with 50 µmol (4 eqv) of N-F-moc-protected amino acid, 50 µmol (4 eqv) of benzotriazole-1-yl-oxy-tris-pyrolidino-phosphonium hexafluorophosphate (PyBop) reagent, and 100 µmol (8 eqv) of N-methylmorpholine were all dissolved in dimethyl formamide. The peptide was cleaved from the polymer by reacting (2 h, 22°C) the resin with TFA/H2O/triethylsilane (90/5/5, v/v/v). The solution containing the crude unprotected peptides was then cooled down to 4°C, precipitated with ether (4°C), and centrifuged (15 min, 3000 rpm, 4°C). The pellet was washed and centrifuged (x3) with ether, dissolved in 30% acetonitrile in H2O, and lyophilized. The lyophilized material was reconstituted in double distilled water before use; only the stock solution, not the diluted material, was stored at -20°C.
Staining of actin cytoskeleton
T cells were incubated (18 h, 37°C, 7.5% CO2, humidified atmosphere) in culture medium. IL-2 was added to the cell cultures, which were then incubated for 48 h. The T cells were then washed and seeded onto FN-covered coverslips in the presence of either PMA (50 ng/ml), IL-2 (100 U/ml), or IL-2 peptides (0.1 ng/ml). After 1 h at 37°C, the adherent cells were fixed (3 min) with paraformaldehyde (3%) and Triton X-100 (0.5%), washed, and fixed (20 min) again with paraformaldehyde (3%). The fixed adherent cells were washed, treated with TRITC-phalloidin, and washed again. Photographs (x1000 magnification) were then taken.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Some cytokines induce adhesion of leukocytes to endothelial
cells, the underlying basement membrane, and to the ECM (3, 4, 6, 20, 21). Therefore, we examined the ability of IL-2 to induce adhesion of
human T cells to ECM, FN, LN, and CO-IV. The results indicated that
IL-2 induced T cell adhesion to FN, LN, and CO-IV (Fig. 1
A), as well as to intact ECM
(Fig. 1B). Note that the adhesion of T cells to LN induced
by IL-2 was lower than that induced by the other ECM
glycoproteins. When T cells were activated only with PMA, 45 ±
4.4% of them adhered to immobilized ECM and ECM glycoproteins (not
shown). IL-2-induced T cell adhesion to the ECM glycoproteins was
inhibited by anti-human ß1 integrin mAbs (not shown),
which suggests that the proadhesive effects of IL-2 were induced via
cell surface-expressed integrins. However, under our experimental
conditions, IL-2 did not alter the T cell surface expression of
ß1 integrins (not shown). Thus, IL-2, in addition to
other proinflammatory mediators, appears to regulate the adhesiveness
of resting human T cells to immobilized ECM and ECM glycoproteins.
|
We have assumed that the degradation of IL-2 can occur in the
inflamed milieu in which both cytokines, such as IL-2, and proteolytic
enzymes, such as neutrophil elastase, are present. We also hypothesized
that, in contrast to the intact IL-2 molecule, certain portions of IL-2
can abrogate the adhesiveness of activated T cells to ECM ligands.
Hence, elastase and soluble IL-2 were incubated together at physiologic
conditions. HPLC analysis of the elastase-degraded IL-2 revealed at
least eight peaks of IL-2, each of which represented at least one low
m.w. protein fragment (Fig. 2).
|
).
Thus, certain IL-2 fragments, obtained by neutrophil
elastase-processing of the cytokine, can inhibit IL-2-induced adhesion
of T cells to FN.
|
Next, the primary sequence of fractions 2 and 8 were analyzed by
gas phase chromatography, because these elastase-generated fractions of
IL-2 appeared to contain adhesion-suppressive peptides. Our analysis
revealed that fragment 2 contained an Ile-Val-Leu (IVL;
IL-2112–114) and an Arg-Met-Leu-Thr (RMLT;
IL-258–61) peptide, whereas fragment 8 contained a
Glu-Phe-Leu-Asn-Arg-Trp-Ile-Thr (EFLNRWIT; IL-2136–143)
octa-peptide. These three peptides were synthesized, and their effects
on IL-2-induced T cell adhesion to FN were studied. The IVL, RMLT, and
EFLNRWIT peptides, inhibited in a dose-dependent manner the adhesion of
IL-2-activated T cells to FN (Fig. 4A). Maximum inhibition was
achieved with 0.1 pg/ml for both IVL, RMLT, and EFLNRWIT (0.29, 0.20,
and 0.09 pM, respectively. The inhibitory dose-response curves of all
peptides are similar to those of the HPLC fractions from which they
were derived.
|
B, indicate that none of these peptides, tested in a broad
range of dosages, interfere with T cell adhesion. Thus, the
anti-adhesive effects of IVL and EFLNRWIT peptides of IL-2 appear
to be due to their direct biologic effect on responding
lymphocytes.
Do the IL-2 peptides, IVL, RMLT, and EFLNRWIT have to be present during
the entire period of the assay to exert their inhibitory effects? The
results, shown in Figure 4C, indicate that most of the
anti-adhesive effects of the three peptides persevered even if
these peptides (at 1 pg/ml) were removed from the T cells before their
activation with IL-2 and seeding onto the FN-coated surfaces.
Apparently, their prolonged inhibitory potential may involve active
intracellular signaling pathways. These results suggest that the IL-2
peptides neither exert their inhibitory activities on T cell adhesion
to FN via binding to the ECM protein, nor to FN-specific
ß1 integrins expressed on the adhering T cells.
IVL, RMLT and EFLNRWIT inhibit T cell adhesion to LN, CO-IV and FN
The next experiment was designed and performed to verify that the
three IL-2-derived peptides indeed affect T cell interactions with ECM
glycoproteins other than FN. T cells were pre-exposed to the three
peptides (at 10 pg/ml), and then activated with IL-2. The treated cells
were then added to microtiter wells coated with CO-IV, LN, and FN. The
results, shown in Figure 5, indicate that
both IVL, RMLT, and EFLNRWIT inhibit T cell adhesion to the three major
cell-adhesive glycoproteins of the ECM (Fig. 5), suggesting that the
elastase-generated IL-2 peptides exert their inhibitory effects over
different subsets of ß1 integrins.
|
Immune cell migration is the outcome of a subtle biologic
equilibrium existing between adhesion and detachment events. Lymphocyte
adhesion to the subendothelial ECM and subsequent migration are two
active processes that can overlap, but are not mutually dependent
events. Adhesion and migration may depend on the ability of the T cells
to continuously integrate different pro- and anti-adhesive signals
via their versatile receptors for ECM, chemokines, cytokines, and
possibly, also antigenic moieties (1, 2). In fact, it has been recently
shown that transient (i.e., low affinity) rather than prolonged
interactions between integrins and the ECM favor IL-4-induced B cell
migration, and not adhesion (28). Therefore, we next examined the
effects of the IVL, RMLT, and EFLNRWIT peptides (at 1 pg/ml) on the
IL-2- and MIP-1ß-induced T cell chemotaxis through FN-coated
polycarbonate membranes. The gradient generated by MIP-1ß and IL-2,
which were placed in the lower compartment of the 48-well chemotaxis
apparatus, induced a marked T cell migration through FN-coated
membranes, which was about 3 to 4-fold higher than the control (Fig. 6). Both IVL, RMLT, and EFLNRWIT markedly
(p < 0.01) inhibited T cell migration toward
IL-2, by about 30, 90, and 60%, respectively. However, although the
IVL peptide, and to a lesser degree also the RMLT peptide, markedly
inhibited (80% and 60%, respectively) T cell chemotaxis toward
MIP-1ß, the EFLNRWIT showed only a limited inhibitory effect on the
chemokine-induced T cell chemotaxis. Thus, in addition to the capacity
of the IVL and EFLNRWIT peptides to inhibit T cell-ECM adhesion, they
seem to inhibit T cell migration through FN in response to a diffusible
gradient produced by IL-2 or MIP-1ß.
|
The preceding chemotaxis experiments indicated that the IVL and
EFLNRWIT peptides can inhibit T cell adhesion and migration through FN
barriers induced not only by IL-2, but also by the chemokine MIP-1ß.
Therefore, in an attempt to further understand the possible physiologic
relevance of such phenomena, we examined the ability of these peptides
to inhibit the adhesion to FN of T cells stimulated by modes other than
IL-2. T cell adhesion to FN can be up-regulated by physiologic
activators such as IL-7 (21) and MIP-1ß (20) and via the CD3 complex,
as well as by nonphysiologic stimuli of integrin avidity, such as PMA
(29), and a ß1 integrin-specific activating mAb, 8A2
(24). This mAb up-regulates the endothelial cell and ECM ligand-binding
activities of the 4ß1 and
5ß1 integrins by binding to their cell
surface-expressed sites, thus converting these nonbinding integrins to
their high affinity, ligand-occupying forms (24).
At 10 pg/ml, both EFLNRWIT and IVL inhibited T cell adhesion to FN that
was induced by various stimulators of T cells and modulators of the
ß1 integrin functions tested (Fig. 7). However, at 0.1 pg/ml, EFLNRWIT did
not inhibit PMA- and 8A2-induced adhesion, and the IVL peptide did not
inhibit PMA-, 8A2-, and anti-CD3 mAb-induced T cell adhesion to FN,
which indicates that these modes of activation are less susceptible to
IL-2-derived peptide-induced suppression than IL-2-mediated activation.
In experiments similar to those shown in Figures 7, A
and B, the control peptides (LVI and TIWRNLFE) did not
affect T cell adhesion to FN induced by the indicated activators (data
not shown). Hence, EFLNRWIT and IVL apparently inhibit T cell adhesion
to the FN component of ECM via a common intracellular event that is
linked to the regulation of the avidities and affinities of
ß1 integrins, and therefore, to their ligand recognition
and binding.
|
The adhesion of immune cells to ECM is dependent on the
sequestering of the cytoplasmic domains of integrins in focal
adhesion sites together with actin-containing microfilament bundles
(30, 31, 32). Therefore, we examined the effect of EFLNRWIT on the
morphologies of adherent T cells. The T cells were activated with IL-2
or PMA, treated with the IL-2 peptides, and seeded onto FN-coated
coverslips. After incubation and fixation, the actin
cytoskeleton of attached T cells was stained with
TRITC-conjugated phalloidin. The morphologies of FN-bound IL-2- and
PMA-activated T cells (Fig. 8,
A and B) were markedly different from those of
nonactivated lymphocytes (Fig. 8E); the activated T cells
appeared spread, and their actin cytoskeleton performed distinct
structures typical of ECM-adherent cells. The EFLNRWIT peptide
inhibited the redistribution of the actin skeleton in both the IL-2-
(Fig. 8C) and PMA-treated (Fig. 8D) FN-adherent T
cells. Control peptides (LVI, TLMR, and TIWRNLFE) did not inhibit the
actin reorganization of the activated T cells (not shown). Hence, the
adhesion-inhibiting activity of the IL-2-derived peptide EFLNRWIT,
similar to the IVL peptide (data not shown), appears to involve
inhibition of the redistribution of the actin cytoskeleton, and
therefore, changes in cell shape and spreading.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
We therefore hypothesized that, in contrast to the proadhesive effects
of the intact IL-2 molecule, certain short IL-2-derived peptides, which
may occur in vivo, can inhibit the interactions of T cells with ECM and
that this interference is independent of the effects of the peptides on
the activation of T cells by IL-2. We also assumed that such moieties
of IL-2 can prevent the arrival of T cells at inflamed sites.
Neutrophil elastase was a likely candidate for the physiologic
production of these inhibitory peptides. Elastase, which is
enzymatically versatile (17, 18), can be expressed and secreted by the
migrating T cells themselves (16) and can transmit mitogenic
stimulations from the environment into the responding T cells (15). In
fact, LPS- and FMLP-activated neutrophils express catalytically active
membrane-bound elastases, proteinase 3, and cathepsin G on their cell
surfaces, which ensures the presence of these enzymes at the leading
edge of the tissue-invading cells (34, 35). Other matrix-degrading
enzymes, such as matrix metalloproteinases, cathepsin B, the
urokinase-type plasminogen activator, and plasmin, can also be bound on
the cell surfaces of fibroblasts and migrating cells (5, 36). An
important feature of elastase is its ability to act in both soluble and
immobilized forms, since a migrating immune cell that expresses
immobilized elastase may encounter matrix-bound IL-2, among other
cytokines. We have found that the processing of rIL-2 by elastase
results in the production of at least eight different products.
Three of these products (present in HPLC fractions 2, 7, and 8)
inhibited IL-2-mediated T cell adhesion to FN. Amino acid composition
analysis and amino acid sequencing revealed that fraction 2
contained the tripeptide IVL (IL-2112–114) and the
tetrapeptide RMLT (IL-258–61), and fraction 8 the
octapeptide EFLNRWIT (IL-2136–143). The RMLT
peptide appeared to be located within the IL-2-binding site of the
-chain of the IL-2R, whereas the IVL and EFLNRWIT are located at
sites far from the receptor-binding sites of IL-2 (7, 8). These
peptides, at a picomolar range of concentrations (i.e., 0.01–1
pg/ml), inhibited the IL-2, as well as MIP-1ß-induced chemotaxis of
human T cells through FN-coated polycarbonate membranes.
The chemoattractive capacity of IL-2 in T cell migration studies in
vitro has been shown using bare polycarbonate filters or
collagen- or Matrigel-coated membranes as immobilized
substrates. T cell migration in these systems was proved to be IL-2R
ß-chain-specific and dependent on the activities of the
matrix-degrading gelatinases (10, 37, 38). Here, in addition to their
antimigratory effects, both peptides of IL-2 inhibited T cell adhesion
to FN induced by various physiologic and nonphysiologic stimuli.
Nevertheless, neither IVL, RMLT, nor EFLNRWIT, at 1 to 100 pg/ml,
interfered with either PHA- or IL-2-mediated proliferative responses of
human T cells, nor did these peptides inhibit the secretion of
TNF- and IFN- from these proliferating cells (not shown).
Moreover, the inversely synthesized molecules LVI, TLMR, and TIWRNLFE
did not inhibit T cell adhesion to FN. Thus, the migration- and
adhesion-suppressive capabilities of IVL, RMLT, and EFLNRWIT are
specific and are not due to toxic cell death.
How do the elastase-derived IL-2 peptides exert their inhibitory
functions? We have demonstrated that the three peptides do not have to
be present during the entire period of the adhesion assay, since their
antiadhesive effect was apparent even after their removal from the
assay before T cell activation with IL-2. This finding also implies
that the IVL, RMLT, and EFLNRWIT peptides do not function by
interacting with putative cell-adhesive epitopes present on the tested
ECM glycoproteins. The existence and activity of putative T cell
surface-expressed receptors specific to the IL-2 peptides described
here require additional study. However, it seems to be highly unlikely
that two of these peptides (IVL and EFLNRWIT) exert their biologic
functions by interacting with the IL-2R subunits or by directly binding
to ß1-specific integrins. The IL-2 peptides interfered
with different modes of T cell activation, leading to their adhesion to
the tested matrix proteins. Moreover, both peptides, used in a
picomolar range of concentrations, appear to block T cell
adhesion not only to FN, mediated predominantly via the
4ß1 and 5ß1
integrins, but also to LN; T cell adhesion to LN was mediated primarily
through the 3ß1 and
6ß1 integrins. Interesting, however, the
RMLT peptide resides within the IL-2R-binding site of IL-2; two
residues, Arg58 and Phe62, which are present
within and adjacent to this peptide, respectively, were shown to be
critical for IL-2-IL-2R interactions (13, 39). Therefore, it will also
be interesting to examine whether the degradation of IL-2 by elastase
produces compounds that can interfere with IL-2 binding to its receptor
and with the biologic outcome of such molecular interactions.
The ability of EFLNRWIT (as well as the other two IL-2 peptides, which
are not shown) to inhibit PMA- and IL-2-induced T cell adhesion to FN
is probably linked to its ability to block the reorganization of the
intracellular actin cytoskeleton. Integrin-cytoskeleton associations
can modulate cell adhesion to ECM ligands, cell spreading in areas of
cell contact with the substratum, and the microclustering and
redistribution of ß1 integrins on the cell surface at
sites of focal adhesion located at the ends of the actin fibers (29, 31, 40). In fact, actin cytoskeleton reorganization, occurring
after activation of leukocytes via a 
-dependent activation of the
isoform of protein kinase C (41, 42), is linked to leukocyte
emigration. Similar observations were noted for T cell activation by
IL-2 (and PMA) and attachment to FN. Although the intracellular
mechanisms of action of the elastase-generated fractions and peptides
have not yet been determined, we postulate that these proteins effect
the adhesion and migration of T cells within the ECM by active
inhibition of intracellular signal transduction pathways linked to
cytoskeleton organization, resulting in an inhibition of
microclustering and an association of integrins with cytoskeletal
elements.
Our findings imply that the tissue-invading T cells themselves can dynamically regulate their own functions. Both adhesion- and migration-promoting stimuli (i.e., intact IL-2) and suppressive by-products of inflammatory mediators can be present, although not necessarily simultaneously, within the inflammatory milieu (43). At the early stages of inflammation, both IL-2 and elastase may function concomitantly to activate T cells to penetrate tissues. Later, the degraded peptide products of IL-2, generated by elastase, may inhibit T cell migration, inhibit the costimulatory effects of IL-2 and other mediators, and probably signal the termination of the inflammatory reaction.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. Ofer Lider, Department of Immunology, Weizmann Institute of Science, Rehovot 76100, Israel. E-mail address:
3 Abbreviations used in this paper: ECM, extracellular matrix; CO-IV, collagen type IV; FN, fibronectin; LN, laminin; MIP-1ß, macrophage inflammatory protein 1ß; TFA, trifluoroacetic acid; TRITC, tetramethyl rhodamine isothiocyanate.
Received for publication February 3, 1998. Accepted for publication April 27, 1998.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Mß2) and modulates polymorphonuclear leukocyte adhesion. J. Exp. Med. 84:1213.
-chain (p55) of the interleukin 2 receptor. Proc. Natl. Acad. Sci. USA 88:4636.-actinin and the ß1 integrin subunit in vitro. J. Cell Biol. 111:721.-like protein family, phosphatidylinositol 3-kinase, and protein kinase C-. J. Immunol. 158:1516.[Abstract]
This article has been cited by other articles:
|
|
 |
|
  U. Sela, N. Mauermann, R. Hershkoviz, H. Zinger, M. Dayan, L. Cahalon, J. P. Liu, E. Mozes, and O. Lider The Inhibition of Autoreactive T Cell Functions by a Peptide Based on the CDR1 of an Anti-DNA Autoantibody Is via TGF-{beta}-Mediated Suppression of LFA-1 and CD44 Expression and Function J. Immunol., December 1, 2005; 175(11): 7255 - 7263. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
U. Sela, R. Hershkoviz, L. Cahalon, O. Lider, and E. Mozes Down-Regulation of Stromal Cell-Derived Factor-1{alpha}-Induced T Cell Chemotaxis by a Peptide Based on the Complementarity-Determining Region 1 of an Anti-DNA Autoantibody via Up-Regulation of TGF-{beta} Secretion J. Immunol., January 1, 2005; 174(1): 302 - 309. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Kipnis, M. Cardon, H. Avidan, G. M. Lewitus, S. Mordechay, A. Rolls, Y. Shani, and M. Schwartz Dopamine, through the Extracellular Signal-Regulated Kinase Pathway, Downregulates CD4+CD25+ Regulatory T-Cell Activity: Implications for Neurodegeneration J. Neurosci., July 7, 2004; 24(27): 6133 - 6143. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
I. Sotnikov, R. Hershkoviz, V. Grabovsky, N. Ilan, L. Cahalon, I. Vlodavsky, R. Alon, and O. Lider Enzymatically Quiescent Heparanase Augments T Cell Interactions with VCAM-1 and Extracellular Matrix Components under Versatile Dynamic Contexts J. Immunol., May 1, 2004; 172(9): 5185 - 5193. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. R. D. Carter, K. M. Whitmore, and R. Thorpe The significance of carbohydrates on G-CSF: differential sensitivity of G-CSFs to human neutrophil elastase degradation J. Leukoc. Biol., March 1, 2004; 75(3): 515 - 522. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Zanin-Zhorov, R. Hershkoviz, I. Hecht, L. Cahalon, and O. Lider Fibronectin-Associated Fas Ligand Rapidly Induces Opposing and Time-Dependent Effects on the Activation and Apoptosis of T Cells J. Immunol., December 1, 2003; 171(11): 5882 - 5889. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Sasaki, T. Tsuji, T. Jinushi, J. Matsuzaki, T. Sato, K. Chamoto, Y. Togashi, T. Koda, and T. Nishimura Differential regulation of VLA-2 expression on Th1 and Th2 cells: a novel marker for the classification of Th subsets Int. Immunol., June 1, 2003; 15(6): 701 - 710. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. Nemoto, H. Tada, and H. Shimauchi Disruption of CD40/CD40 ligand interaction with cleavage of CD40 on human gingival fibroblasts by human leukocyte elastase resulting in down-regulation of chemokine production J. Leukoc. Biol., September 1, 2002; 72(3): 538 - 545. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Ariel, D. Novick, M. Rubinstein, C. A. Dinarello, O. Lider, and R. Hershkoviz IL-12 and IL-18 induce MAP kinase-dependent adhesion of T cells to extracellular matrix components J. Leukoc. Biol., July 1, 2002; 72(1): 192 - 198. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Iwadate, A. Yamaura, Y. Sato, S. Sakiyama, and M. Tagawa Induction of Immunity in Peripheral Tissues Combined with Intracerebral Transplantation of Interleukin-2-producing Cells Eliminates Established Brain Tumors Cancer Res., December 1, 2001; 61(24): 8769 - 8774. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. De Noncourt, O. Robledo, T. Alain, A. E. Kossakowska, S. J. Urbanski, E. F. Potworowski, and Y. St-Pierre Leukocyte elastase in murine and human non-Hodgkin lymphomas J. Leukoc. Biol., October 1, 2001; 70(4): 585 - 591. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 W. L. LEE and G. P. DOWNEY Leukocyte Elastase . Physiological Functions and Role in Acute Lung Injury Am. J. Respir. Crit. Care Med., September 1, 2001; 164(5): 896 - 904. [Full Text] [PDF]
|
|
|
|
|
|
A. Brill, R. Hershkoviz, G. G. Vaday, Y. Chowers, and O. Lider Augmentation of RANTES-Induced Extracellular Signal-Regulated Kinase Mediated Signaling and T Cell Adhesion by Elastase-Treated Fibronectin J. Immunol., June 15, 2001; 166(12): 7121 - 7127. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Girard, P. Cléroux, P. Tremblay, S. Dea, and Y. St-Pierre Increased proteolytic activity and matrix metalloprotease expression in lungs during infection by porcine reproductive and respiratory syndrome virus J. Gen. Virol., June 1, 2001; 82(6): 1253 - 1261. [Abstract] [Full Text]
|
|
|
|
|
|
G. G. Vaday, S. Franitza, H. Schor, I. Hecht, A. Brill, L. Cahalon, R. Hershkoviz, and O. Lider Combinatorial signals by inflammatory cytokines and chemokines mediate leukocyte interactions with extracellular matrix J. Leukoc. Biol., June 1, 2001; 69(6): 885 - 892. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 O. Kollet, A. Spiegel, A. Peled, I. Petit, T. Byk, R. Hershkoviz, E. Guetta, G. Barkai, A. Nagler, and T. Lapidot Rapid and efficient homing of human CD34+CD38{-}/lowCXCR4+ stem and progenitor cells to the bone marrow and spleen of NOD/SCID and NOD/SCID/B2mnull mice Blood, May 15, 2001; 97(10): 3283 - 3291. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Ariel, R. Hershkoviz, I. Altbaum-Weiss, S. Ganor, and O. Lider Cell Surface-Expressed Moesin-Like Receptor Regulates T Cell Interactions with Tissue Components and Binds an Adhesion-Modulating IL-2 Peptide Generated by Elastase J. Immunol., March 1, 2001; 166(5): 3052 - 3060. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
U. Bank and S. Ansorge More than destructive: neutrophil-derived serine proteases in cytokine bioactivity control J. Leukoc. Biol., February 1, 2001; 69(2): 197 - 206. [Abstract] [Full Text]
|
|
|
|
|
|
S. Franitza, R. Hershkoviz, N.'a. Kam, N. Lichtenstein, G. G. Vaday, R. Alon, and O. Lider TNF-{alpha} Associated with Extracellular Matrix Fibronectin Provides a Stop Signal for Chemotactically Migrating T Cells J. Immunol., September 1, 2000; 165(5): 2738 - 2747. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
