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1
*
Department of Medicine, Meir General Hospital, Kfar-Saba, Israel;
Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel; and
Department of Immunology, Weizmann Institute of Science, Rehovot, Israel
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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upon direct contact with activated T cells. This
data suggested an unrecognized activation pathway, where mast cells may
be activated during T cell-mediated inflammation. Herein, we show that
this cell-cell contact results in the release of matrix
metalloproteinase (MMP)-9 and the MMP inhibitor tissue inhibitor of
metalloproteinase 1 from HMC-1 human mast cells or from mature
peripheral blood-derived human mast cells. The expression and release
of these mediators, as well as of 
-hexosaminidase and several
cytokines, were also induced when mast cells were incubated with cell
membranes isolated from activated, but not resting, T cells.
Subcellular fractionation revealed that the mature form of MMP-9
cofractionated with histamine and tryptase, indicating its localization
within the secretory granules. MMP-9 release was first detected at
6 h and peaked at 22 h of incubation with activated T cell
membranes, while TNF- release peaked after only 6 h.
Anti-TNF- mAb inhibited the T cell membrane-induced MMP-9 release,
indicating a possible autocrine regulation of MMP release by mast cell
TNF-. This cascade of events, whereby mast cells are activated by T
cells to release cytokines and MMP-9, which are known to be essential
for leukocyte extravasation and recruitment to affected sites, points
to an important immunoregulatory function of mast cells within the
context of T cell-mediated inflammatory
processes. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Morphologic studies have revealed that mast cells reside in close apposition to T cells within inflamed allergic tissues and at sites of parasitic infections (4, 5). Hence, a functional relationship between these two immune cell types has been proposed (6). Consistent with this hypothesis, it has been demonstrated that the mononuclear cell infiltration in the late allergic response is mast cell dependent (7). Studies have also shown that mast cells are activated during T cell-mediated inflammatory reactions, such as cutaneous delayed hypersensitivity (8), rheumatoid arthritis (6), and hypersensitivity pneumonitis (9). It seems likely that such a relationship may provide bidirectional signals, since T cells can mediate mast cell proliferation and activation (6, 10, 11), and, on the other hand, mediators of activated mast cells may affect T cell-mediated inflammatory reactions (6, 12). Thus, intercellular communication via cell-cell interactions is likely a significant mechanism of immune cell functions at inflamed sites, where several cell types tend to accumulate (13).
We have previously reported on the effects of direct contact between
mast cells and T lymphocytes on mast cell degranulation. Mast cells
were found to degranulate in response to direct contact with activated
T cells as well as to produce TNF- (14). Moreover,
studies using murine mast cells and PMA- or anti CD3-activated T cells
attributed the degranulation induced by cell-cell contact to
ICAM-1-LFA-1 interactions (15), thus providing further
evidence of a putative relationship between the two cell types.
Mast cell function in potentiating multiple responses in their resident
tissues, via the release of soluble mediators, may depend on
interactions with extracellular matrix
(ECM)4 molecules
within connective tissues. Several studies have highlighted the
potential role of mast cells in mediating ECM degradation through the
activation and production of matrix metalloproteinases (MMPs). Mast
cell tryptase and chymase have been shown to activate the precursors of
MMP-2 (gelatinase A) (16), MMP-9 (gelatinase B)
(17), collagenase, and stromelysin (18).
Recently, it has been demonstrated that activation of mast cells by
long (48 h) incubation with PMA stimulated MMP-9 expression in vitro.
This enzyme has been identified in mast cells localized in various
healthy and diseased tissues (19). Reports of differential
regulation by the c-kit ligand stem cell factor (SCF), a
potent stimulator of mast cell proliferation and differentiation, and
the pleiotrophic cytokine TGF- implicate the importance of growth
factors in mediating MMP-9 secretion by mast cells from various sources
(20, 21). Although previous studies have indicated a role
for direct cell-cell contact in potentiating metalloproteinase
expression in monocytes (13) and T lymphoma cells
(22), thus far mast cell-T cell contact has not been
reported to induce mast cell production of MMPs.
The current study was aimed at gaining insight into the functional role
of mast cell-T cell contact in expression and release of MMPs, in the
context of modification of the inflammatory environment. We now
demonstrate that mast cell-T cell heterotypic adhesion up-regulates
mast cell MMP-9 expression as well as release of active MMP-9 and
tissue inhibitor of metalloproteinase (TIMP) 1 from granular stores.
Furthermore, although mast cell expression or release of the
inflammatory cytokines TNF-, IL-4, and IL-6 was induced by direct
contact with activated T cells, only TNF- regulated induction of
MMP-9 expression. These results suggest that physical interactions
between mast cells and activated T lymphocytes may promote mast cell
release of soluble cytokines and proteases that regulate ECM
degradation during T cell-mediated inflammation.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The following Abs were purchased from R&D Systems (Minneapolis,
MN): normal mouse IgG1 isotype control, anti-human
IL-4-neutralizing mAb, anti-human IL-6-neutralizing mAb, and
anti-human pro/active MMP-9 mAb. Anti-TNF--neutralizing mAb was
purchased from BD PharMingen (San Diego, CA). Anti-human cathepsin D Ab
was purchased from Santa Cruz Biotechnology (Santa Cruz, CA).
Anti-human mast cell tryptase mAb was purchased from Chemicon
(Temecula, CA).
Cells
Reagents for cell culture were purchased from Biological Industries (Beit Haemek, Israel). HMC-1 cells (23), a human mast cell leukemia cell line, and the Jurkat T cell lymphoma line were each maintained in RPMI 1640 supplemented with 10% FCS, 2 mM L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, and 12.5 U/ml nystatin. Human peripheral blood T lymphocytes were isolated from healthy donors as previously described (12). Briefly, the mononuclear cells were isolated on Ficoll gradients, then washed and incubated for 2 h at 37°C in 5% CO2 in a humidified incubator. Nonadherent cells were collected and incubated on nylon wool columns. Unbound cells were eluted by extensive washing with PBS, and the resulting cell population was >98% T lymphocytes.
To study the effects of coculture with T cells, freshly isolated human peripheral blood T lymphocytes or Jurkat T cells (1 x 106/ml) were first activated with 50 ng/ml PMA for 60 min at 37°C, followed by extensive washing (three times) with RPMI 1640. Mast cells (1 x 106/ml) were then cocultured for 20–22 h with activated or nonactivated T cells (at 1:1 ratio) in RPMI 1640 supplemented with L-glutamine, penicillin-streptomycin-nystatin, and 0.1% BSA in a humidified incubator in 96- or 24-well culture plates.
Human peripheral blood CD34+ progenitor cells were isolated as described elsewhere (24), then cultured in serum-free medium (StemPro-34 SFM; Life Technologies, Grand Island, NY) supplemented with L-glutamine (200 mM), streptomycin (50 mg/ml), penicillin (100 U/ml), recombinant human IL-6 (100 ng/ml), human SCF (100 ng/ml; PeproTech, Rocky Hill, NJ), and 30 ng/ml rhIL-3 (for the first week only). Half of the culture medium was replaced with fresh medium every 7 days. Purity of mast cells was determined by metachromatic staining using acidic toluidine blue (pH 1.0). After 8–10 wk of establishing the culture, >95% of the cells were identified as mast cells. Contaminating monocytes/macrophages were removed by incubation on plastic culture dishes (35 x 10 mm) for 2 h, finally yielding a >99% mast cell population.
Preparation of T cell membranes
Two methods for isolation of Jurkat T cell membranes were used. Method 1 is a modification of a method described previously (25). Resting or PMA-activated cells (2 x 108) were washed three times with PBS (8 min, 150 x g) and resuspended at 107 cells/ml in ice-cold TKMS lysis buffer comprised of 50 mM Tris-HCl (pH 7.4), 25 mM KCl, 5 mM MgCl2, 0.25 M sucrose, 1 mM PMSF, and Complete, a mixture of protease inhibitors (Boehringer Mannheim, Mannheim, Germany). The cells were kept on ice for 20 min and lysed by five cycles of freezing and thawing in liquid nitrogen until no living cells could be observed by trypan blue exclusion. The cells were then centrifuged at 800 x g for 5 min at 4°C. The supernatants were collected and subjected to centrifugation for 60 min at 100,000 x g at 4°C. The pellets were suspended in PBS and stored at -70°C. Method 2 is a modification of a method described previously (26). Briefly, resting or PMA-activated cells (2 x 108) were washed with PBS as above and resuspended at 107 cells/ml in ice-cold STM lysis buffer comprised of 0.25 M sucrose, 5 mM Tris-HCl (pH 7.2), and 1 mM MgCl2. PMSF and Complete protease inhibitors were added. The cells were kept on ice for 10 min and lysed by two cycles of freezing and thawing in liquid nitrogen. The cell lysates were then centrifuged at 280 x g for 5 min at 4°C. The supernatants were collected and centrifuged at 1500 x g for 10 min at 4°C. The pellets were suspended in 9 ml of 1.42 M sucrose-STM, overlaid with 2 ml of 0.25 M sucrose-STM, and centrifuged for 60 min at 82,000 x g (SW41 rotor). The interphases between the two sucrose layers were collected and 8 ml of 5 mM Tris-HCl (pH 7.2) were added. These suspensions were further centrifuged for 60 min at 100,000 x g. The final pellets were suspended in PBS and stored at -70°C.
Gelatin zymography
Supernatants of mast cells, T cells, cocultures of both cell types, or mast cells incubated with T cell membranes were analyzed by gelatin zymography to detect gelatinase activity. Aliquots (20 µl) of cell supernatants were subjected to electrophoresis under nonreducing conditions in 10% polyacrylamide gels containing 1 mg/ml gelatin type A (Sigma, St. Louis, MO). Gels were washed three times in 2.5% Triton X-100 to renature the gelatinases, then incubated overnight in 50 mM Tris-HCl (pH 7.5) and 5 mM CaCl2. Coomassie blue staining, followed by destaining, allowed visualization of clear zones of lysis against a blue background.
TIMP-1 ELISA
Supernatants of HMC-1 cell cultures were tested for levels of secreted TIMP-1 using a commercial Biotrak ELISA system according to the manufacturer’s instructions (Amersham Pharmacia Biotech, Buckinghamshire, U.K.).
-Hexosaminidase release
Activity of the secretory granule-associated enzyme
-hexosaminidase was determined by incubating 20-µl aliquots of
supernatants and cell lysates for 90 min at 37°C with 50 µl of
substrate solution consisting of 1.3 mg/ml
p-nitrophenyl-N-acetyl--D-glucosaminide
(Sigma) in 0.1 M citrate (pH 4.5). Reactions were stopped by the
addition of 150 µl of 0.2 M glycine (pH 10.7). OD was read at 405 nm
using an ELISA reader. Results (mean ± SD) were expressed as
percentage of total -hexosaminidase activity present in the
cells.
TNF- bioassay
Released TNF- was measured in supernatants of HMC-1 cell
cultures as previously described (14). Briefly, the
supernatants were added to cultures of the TNF--sensitive mouse
fibrosarcoma cell line L-929. Cell death, caused by TNF- in the
HMC-1 cell supernatants, was quantified by comparison with titration
curves of cell death due to the addition of purified TNF-
(PeproTech).
Subcellular fractionation of HMC-1 cells
Fractionation of HMC-1 cells was performed essentially as described elsewhere (27). HMC-1 cells were washed with PBS and resuspended in homogenization buffer comprised of 0.25 M sucrose, 1 mM MgCl2, 800 U/ml DNase I (Sigma), 10 mM HEPES (pH 7.4), 1 mM PMSF, and a protease inhibitor mixture (Complete). Cells were then disrupted by five cycles of freezing and thawing. Unbroken cells and nuclei were removed by sequential filtering through 5- and 2-µm pore size filters (Poretics, Livermore, CA). The final filtrate was centrifuged for 10 min at 500 x g, and the supernatant was loaded onto a continuous 0.45–2.0 M sucrose gradient (10 ml) and centrifuged for 18 h at 100,000 x g. Thirty fractions were taken from the top of the gradient.
Histamine assay
Histamine content in the gradient fractions was assayed fluorometrically after condensation in alkaline medium with o-phthaldialdehyde (28).
SDS-PAGE and immunoblotting
Samples were separated by SDS-PAGE using 10% polyacrylamide gels and transferred to nitrocellulose filters. Blots were blocked for 3 h in TBST (10 mM Tris-HCl (pH 8.0), 150 mM NaCl, and 0.05% Tween 20) containing 5% skim milk, followed by overnight incubation at 4°C with the indicated primary Abs. Blots were washed three times and incubated for 1 h at room temperature with HRP-conjugated secondary Ab. Immunoreactive bands were visualized using the ECL method according to standard procedures.
Cathepsin D content
Aliquots of the gradient fractions were mixed with 5x Laemmli sample buffer, boiled for 5 min, and subjected to SDS-PAGE and immunoblotting. Blots were processed with anti-cathepsin D Abs. Results were expressed as percentage of total OD as determined by densitometry of the immunoreactive bands using the TINA-PCbas software (Ray test, Isotopenmessgeräte, Germany) (27).
Semiquantitative RT-PCR
HMC-1 cells were incubated for 20 h with cellular membranes isolated from resting or activated Jurkat T cells, then lysed in TRIzol reagent (Life Technologies, Rockville, MD). Total RNA was isolated according to the manufacturer’s instructions, and 30 µg was treated with DNase I (Amersham Pharmacia Biotech). RNA samples (5 µg) were reverse transcribed using 20 U avian myeloblastosis virus-reverse transcriptase (Promega, Madison, WI) or 20 U of SuperScript II (Life Technologies) and amplified using specific sets of primers. The linear ranges of PCR amplification were determined for each transcript, and amplification was performed within those linear ranges. PCR samples were subjected to electrophoresis on 1% agarose gels.
The following oligonucleotide primers were synthesized at the Nucleotide Core Facility (Weizmann Institute of Science, Rehovot, Israel): human MMP-9 sense, 5'-GACTCTACACCCGGGACGGCAATGCTG; human MMP-9 antisense, 5'-CGTCCACCGGACTCAAAGGCACAGTAG; GAPDH sense, 5'-CGGAGTCAACGGATTTGGTCGTAT; and GAPDH antisense, 5'-AGCCTTCTCCATGGTTGGTGAAGAC.
The following oligonucleotide primers were synthesized by
Life Technologies: human MMP-2 sense, 5'-CTTTTTCAAAGGTGACCGCTAC; human
MMP-2 antisense, 5'-TAGAAGTAGGTGTAGGCTGCGTC; human IL-4 sense,
5'-CAGTTCTACAGCCACCATGAGA; human IL-4 antisense,
5'-CATGATCGTCTTTAGCCTTTCC; human IL-6 sense,
5'-CCAGGAGAAGATTCCAAAGATG; human IL-6 antisense,
5'-GGAAGGTTCAGGTTGTTTTCTG; human TNF- sense,
5'-CTGTACCTCATCTACTCCCAGGTC; human TNF- antisense,
5'-AGACTCGGCAAAGTCGAGATAGT; human TNF- sense,
5'-CTGTACCTCATCTACTCCCAGGTC; and human TNF- antisense,
5'-AGACTCGGCAAAGTCGAGATAGT.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Although mast cell exocytosis and release of serine proteases
critical in inflammation have been extensively studied, only recently
has evidence on the regulation of mast cell MMP synthesis emerged.
Soluble mediators, including SCF and TGF- (20, 21), and
phorbol esters (19) have been shown to modulate expression
of mast cell MMP-9. However, the role of intercellular communication in
modulating such expression, via cell-cell contact, remains
unresolved.
We first determined whether mast cell-T cell interactions, which likely
occur at sites of inflammation, could potentiate mast cell production
of MMP-9. HMC-1 mast cells were incubated overnight with resting or
activated peripheral blood-derived human T cells or Jurkat T cells in
serum-free media; supernatants were then collected and analyzed for
released mediators. Zymographic analysis of supernatants obtained from
single cultures of resting or activated peripheral blood T cells or
Jurkat T cells showed low basal levels of released MMP-9 (by activated
Jurkat T cells) or no release at all (Fig. 1
). Moreover, incubation of mast cells
with resting T cells had no effect on MMP-9 release. In sharp contrast,
coculture with activated peripheral blood-derived human T cells or
Jurkat T cells markedly potentiated MMP-9 release by HMC-1 cells (Fig. 1). Western blotting using anti-pro/active MMP-9 mAb indicated that
the released MMP-9 was exclusively in the active 84-kDa form (data not
shown). A 2- to 3-fold increase in HMC-1 cell degranulation, as
determined by -hexosaminidase activity, was found after coincubation
with activated, but not resting, both T cell types (Fig. 1). These
results suggest that mast cell MMP-9 release is highly regulated by
interactions with activated, but not resting, T cells. Since similar
results were obtained using either peripheral blood T lymphocytes or
Jurkat T cells, subsequent experiments were performed using Jurkat T
cells. As has been previously reported by us (14, 15),
using a microporous membrane (Transwell; Costar, Cambridge, MA)
to separate between the two cell populations, or supernatants from
activated T cells did not induce -hexosaminidase nor MMP-9 release
from HMC-1 mast cells (data not shown).
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-hexosaminidase release was
due to direct cell-cell contact and to enable us to work with a
single-cell system (i.e., mast cells), membranes from resting or
activated Jurkat T cells were isolated and incubated with HMC-1 cells
for 20 h. Membranes obtained from activated, but not resting,
Jurkat T cells induced mast cell degranulation, as demonstrated by a
pronounced increase (
5-fold) in -hexosaminidase activity in the
supernatants (Fig. 2A).
Furthermore, MMP-9 release was markedly induced in HMC-1 cells via
direct contact with activated, but not resting, T cell membranes (Fig. 2B, upper panel). This result was also confirmed
by Western blotting using anti-MMP-9 mAb (Fig. 2B,
lower panel). Similar results, that is, T cell
membrane-induced -hexosaminidase and MMP-9 release (data not shown),
were obtained by using an additional method for isolation of T cell
membranes (method 2 in Materials and Methods), which has
been reported to provide purified plasma membranes with minimal
microsomal/lysosomal contamination (26).
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C). In contrast, MMP-2 was
found to be constitutively expressed in resting and activated HMC-1
cells (Fig. 2C).
Experiments were also performed using mature (9- to 10-wk-old)
peripheral blood (CD34+)-derived primary human
mast cell cultures. Similar to our findings with the HMC-1 cell line, a
marked increase in -hexosaminidase release (>4-fold; data not
shown) was observed as well as a pronounced augmentation in MMP-9
release in response to direct contact of these primary mast cell
cultures with activated T cell membranes (Fig. 2D). These
data provide evidence that direct contact between cell surface
molecules on mast cells and on activated T cell membranes is sufficient
to transduce the stimulatory signal in mast cells necessary for
degranulation and MMP-9 release, independent of T cell intracellular
function or production of cytokines and other mediators.
Kinetics of MMP-9 and -hexosaminidase release
Although both -hexosaminidase and active MMP-9 were secreted
from mast cells upon activation with activated T cell membranes, the
rate of their release was not defined. To compare the kinetics of
-hexosaminidase and active MMP-9 release, HMC-1 cells were incubated
with activated T cell membranes for various periods of time, and
supernatants were collected for analysis of these mediators.
-Hexosaminidase release was first noticed after 4 h of
incubation with activated T cell membranes and increased over time,
sharply peaking at 22 h (Fig. 3).
This very slow pattern of secretion kinetics resembled very much that
of MMP-9, except for a later onset of the latter (6 h as compared with
4 h; Fig. 3). The later onset of secretion may be explained by the
fact that in contrast to MMP-9, -hexosaminidase is prestored in mast
cell granules (29).
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Although enzymatic activity of MMPs within an inflammatory milieu
is partly determined by their levels of expression, their capacity to
degrade ECM is also influenced by the expression of TIMPs, the natural
inhibitors of MMP activity. TIMP-1, which is a prevalent TIMP found in
body fluids, has been shown to be inducible in immune cells by several
factors, including cytokines (30) and phorbol esters
(31). To our knowledge, no studies to date have described
mast cell synthesis of TIMP-1. Thus, we examined whether activation of
HMC-1 cells by T cell membranes exerts a stimulatory effect on mast
cell production of TIMP-1. Secreted TIMP-1 was measured in
supernatants, and the results are presented in Fig. 4. HMC-1 cells alone or HMC-1 cells
incubated with resting T cell membranes secreted low levels (<10
ng/ml) of TIMP-1. In contrast, HMC-1 cells incubated with activated T
cell membranes demonstrated a significant increase in the TIMP-1 level
(120 ng/ml; Fig. 4).
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Considering our finding that MMP-9 release, induced by mast cell-T
cell interactions, corresponded with -hexosaminidase release, it was
important to determine whether MMP-9 was stored and secreted from the
secretory granules of mast cells. MMP-9 gene expression was clearly
induced by such interactions (Fig. 2C), and several studies
have demonstrated induction of MMP-9 mRNA in mast cells from various
species (19, 20, 21); however, no studies to date have
reported whether MMP-9 is transported into, and possibly stored in, the
secretory granules.
To examine the cellular localization of the newly synthesized MMP-9,
HMC-1 cells were incubated with membranes of activated T cells for
20 h, then subjected to subcellular fractionation using continuous
sucrose gradient. Samples of fractions taken from the gradient were
subjected to SDS-PAGE and immunoblotting, and analyzed by using
anti-human MMP-9 mAb that recognizes both the proenzyme and the
active forms of MMP-9. As shown in Fig. 5A, the higher molecular
mass form of MMP-9, corresponding to the proenzyme of 92 kDa,
was predominantly found in fractions 5–10. Densitometric analysis of
the Western blot indicated a distinct peak at fraction 8, with
7-fold more pro-MMP-9 (92 kDa) than active MMP-9 (84 kDa; Fig. 5B). In contrast, a second peak consisting only of active
MMP-9 (84 kDa), the predominant form seen by gelatin zymography and
Western blotting (Figs. 1 and 2), was found in fractions 17–22 (Fig. 5, A and B). These results suggest that the
proenzymes and active enzymes are distributed in distinct subcellular
compartments upon induction of expression and protein synthesis. Of
note, MMP-9 was absent from sucrose gradient fractions of control HMC-1
cells or cells incubated with nonactivated T cell membranes (data not
shown).
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-hexosaminidase, and the lysosomal enzyme cathepsin D in the
gradient fractions was determined. A major peak of the mature form of
cathepsin D was found in fractions 4–10. Yet a smaller peak (15%
of the total cathepsin D) was found in fractions 15–21 (Fig. 5D). -Hexosaminidase was also distributed between these
two peaks, with 60% of the total activity present in fractions 5–12.
Histamine, which is a major constituent of mast cell secretory
granules, migrated particularly at fractions 15–21, along with the
remaining -hexosaminidase activity (Fig. 5D). Histamine
was also found at the top of the gradient, but this probably reflected
the cytosolic pool and the content of secretory granules that were
released during cell disruption. Tryptase, another prototypic
granule-associated enzyme, was present in fractions 16–21 (Fig. 5C). It has previously been reported that fractions
containing very high amounts of cathepsin D and -hexosaminidase are
likely to represent lysosomal/endosomal compartments (fractions 5–10,
Fig. 5C), whereas fractions containing tryptase and
histamine, as well as -hexosaminidase (fractions 15–21; Fig. 5, C and D), likely represent the secretory granule
compartment of HMC-1 cells (27). Taken together, it
appears that the proenzyme of MMP-9 (92 kDa) may be transported to both
lysosomes/endosomes and secretory granule compartments. However, only
at the secretory granules is the enzyme converted to the 84-kDa
active form. Expression of cytokines induced by cell-cell contact
Since certain cytokines are known modulators of MMP production by
leukocytes (31, 32, 33), we sought to investigate whether mast
cell activation by T cell membranes results in cytokine expression.
Therefore, the expression of the inflammatory cytokines IL-4 and IL-6
by mast cells was investigated. IL-4 gene expression was not detected,
by RT-PCR, in control cells or cells incubated with resting T cell
membranes. However, direct contact with cell membranes from activated T
cells induced IL-4 expression (Fig. 6).
Low levels of IL-6 mRNA were detected by RT-PCR in control cells or
cells incubated with resting T cell membranes. Yet, direct contact with
cell membranes from activated T cells markedly increased IL-6
expression (Fig. 6). Thus, MMP-9 gene expression appeared to be
concomitantly induced with that of IL-4 and IL-6.
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release
We have previously reported that in addition to stimulating mast
cell degranulation, activated T lymphocytes also stimulate human mast
cells to secrete TNF- upon heterotypic aggregation
(14). Release of TNF- was likely independent of the
mechanism leading to mast cell degranulation, since the
phosphatidylinositol 3-kinase inhibitor wortmannin blocked
-hexosaminidase release, but not TNF- secretion
(14). TNF- is considered a potent stimulator of MMP-9
release in leukocytes (30, 31, 32). We therefore studied the
kinetics of TNF- secretion by mast cells, induced by activated T
cell membranes, compared with that of MMP-9. As can be seen in Fig. 7, TNF- release started as early as
4 h following the incubation with activated T cell membranes,
reaching a maximum at 6 h. These TNF- kinetics are similar to
those reported for IgE-mediated mast cell activation (34).
In contrast, the release of active MMP-9 started only after 6 h of
incubation, with a prolonged and steady rise to a peak at 22 h of
incubation (Fig. 7). Accordingly, the release of TNF- clearly
precedes MMP-9 release and thus TNF- may be a key factor in the
regulation of MMP-9 expression and release.
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mAb inhibits MMP-9 induction and release
To confirm the dependency of MMP-9 induction on cytokine
production, HMC-1 cells were cultured with activated T cell membranes
for 20 h in the presence or absence of neutralizing mAb against
human TNF-, IL-4, and IL-6. Supernatants were collected for analysis
of MMP-9 secretion and -hexosaminidase release. Abs to TNF-,
IL-4, and IL-6 had no effect on -hexosaminidase release induced by
incubation with T cell membranes (data not shown). On the other hand,
anti-TNF- mAb inhibited both expression and release of MMP-9
induced by activated T cell membranes (Fig. 8, A and C),
whereas anti-IL-4 and anti-IL-6 mAb did not alter levels of
released MMP-9 (Fig. 8B). MMP-2 was unaffected by the Abs
tested (Fig. 8A). Interestingly, experiments using HMC-1
cells incubated with increasing doses (1–20 ng/ml) of purified TNF-
indicated that soluble TNF- alone is not sufficient to induce mast
cell release of MMP-9 (data not shown).
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release. These findings provide further support of a functional
mast cell-T cell relationships. Cell-cell transmission of signals is
likely leading to important mast cell activities in allergic and T
cell-mediated inflammation. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Mast cells and T lymphocytes clearly have bidirectional influences on
each other, as evidenced during T cell-mediated inflammation (6, 8, 9) and parasitic infections (3, 5). Such
influences have primarily been attributed to the biological effects of
T cell-derived soluble mediators on mast cell function (38, 39). However, recent investigations of mast cell-T cell
interactions have revealed a novel intercellular communication
exclusively involving the binding of cell surface molecules. Mast cells
have been shown to degranulate and produce TNF- upon direct contact
with activated T cells (14), a process that may be
regulated by LFA-1-ICAM-1 interaction (15). Considering
that such T cell-dependent mast cell activation may also involve other
alterations in mast cell functions, we undertook the present study to
investigate the role of mast cell-T cell interactions in regulating
mast cell production of MMP-9 and TIMP-1, important mediators of ECM
degradation. We now show that activated T cells cause a marked increase
in the release of MMP-9 from human mast cells (Fig. 1). We further
demonstrate that this effect is mediated by a direct contact of
activated T cell membranes with mast cells (Fig. 2). This effect was
established by using two types of human mast cells: the HMC-1 cell line
(Fig. 2B) and primary cultures of mast cells isolated from
CD34+ peripheral blood cells (Fig. 2D).
Several lines of evidence have implicated cell-cell interactions between inflammatory cells as a mode of either bilaterally or directionally regulating MMP expression via specific receptors and counterreceptors. Endothelial cells can directly up-regulate expression of MMP-9 in monocytes (40) and in T cells (22) via ICAM-1-LFA-1 interactions. T cells are capable of directly inducing MMP-9 expression in fibroblasts (41), neutrophils (42), and monocytes (13, 43) through cell-cell contact. In addition to stimulating monocyte production of MMP-9 (43), interaction between CD40 on monocytes and CD40 ligand (gp39) on T cells was shown to stimulate monocyte expression of various cytokines and adhesion molecules (44).
To our knowledge, the results presented herein are the first evidence
of MMP-9 regulation in mast cells by direct contact with another
leukocyte population. Such contact with T lymphocytes correlated with
induction of degranulation (Figs. 1 and 2A), release of
TNF-, and expression of the cytokines IL-4 and IL-6 (Fig. 6).
Analysis of the kinetics of TNF- production indicated that this
cytokine was released by HMC-1 cells early (40% of maximal release as
soon as 4 h of coincubation, reaching a maximum at 6 h) in
response to interaction with activated T cell membranes (Fig. 7). In
contrast, MMP-9 release required extended periods of incubation (22 h)
with T cell membranes before a peak of secretion was attained (Figs. 3 and 7). These very slow kinetics of MMP-9 release appears to be
correlated with that of the secretory granule exocytosis, as indicated
by secretion of the granule associated enzyme -hexosaminidase (Fig. 3). It seems therefore plausible that TNF- may be an early mediator
that is produced in response to cell-cell contact, thereby leading to
MMP-9 expression. The newly released TNF- may be specifically
involved in the up-regulation of MMP-9 expression, but not in the
secretion of -hexosaminidase. Thus, these findings further support
the notion that TNF- may be required early for an autocrine
regulatory pathway of MMP-9 gene expression, but not for the exocytotic
degranulation process. TNF- has been shown to be a potent stimulator
of MMP-9 production (31, 32). However, although
neutralization of TNF- during incubation of mast cells with
activated T cell membranes blocked MMP-9 production (Fig. 8), exogenous
TNF- was not sufficient to stimulate HMC-1 cell expression of MMP-9
(data not shown). This may be ascribed to a need for a preactivation
state of the mast cells, which is achieved by incubation with activated
T cells or T cell membranes and which is absent when the cells are
incubated with soluble TNF- alone.
Recently, the regulation of mast cell MMP activation by endogenous
proteinases has been investigated. Dog mastocytoma cells were shown to
constitutively secrete the zymogen and the active forms of MMP-9 and
the active form of MMP-2. Endogenous -chymase, released upon
degranulation, was shown to be responsible for the activation of these
MMP enzymes (17). Activation of MMP-9 and MMP-2 has also
been attributed to the dog mastocytoma cell MMP-3, which is
preactivated with endogenous tryptase or chymase (18). Our
data on the subcellular distribution of MMP-9 in HMC-1 cells upon
incubation with activated T cell membranes (Fig. 5) suggest that the
mechanism of cell-cell stimulation plays a major role in the release of
active MMP-9 from within mast cell granules. Such localization of MMP-9
in the granular fractions of human mast cells, predominantly as an
active enzyme (84 kDa), is a novel finding, since previous studies only
demonstrated activation of MMP-9 by extracellular -chymase
(17). As illustrated in Fig. 9, it is conceivable from the data
presented herein that upon contact with activated T cells, mast cell
expression of MMP-9 is up-regulated by either endogenous soluble
TNF- or T cell membrane-bound TNF-. The newly synthesized zymogen
(92 kDa) is then transported into endosomes/lysosomes and into the
secretory granules as well. -Chymase, tryptase, or other proteinases
prestored at the secretory granules may convert the zymogen into its
active 84-kDa form. This model of intracellular MMP-9
localization/activation is further supported by the observations that
1) direct contact with activated T cell membranes concomitantly
stimulates mast cell degranulation in similar time kinetics as MMP-9
release and 2) active MMP-9 cofractionated with histamine and tryptase
in the granular fraction of subcellular components. Thus, we propose
that mast cells may receive stimulatory signals while interacting with
activated T lymphocytes, that may serve to influence local MMP-9
release, as well as other mast cell activities within the inflammatory
microenvironment. We currently focus on identifying the specific
stimulatory signals expressed by activated T cells that lead to
enhancement of MMP synthesis and release.
|
), in regulating the activity
of MMP-9 is not yet evident. Correlative up-regulation of MMP and
TIMP-1 release by cytokines or other stimuli has been described in
other leukocytes (30, 31). Considering the robust
responses of mast cells to direct contact with activated T cells, it is
probable that TIMP-1 is also induced to counterbalance the substantial
levels of active MMP-9 released from secretory granules. This form of
regulation may aid in limiting and defining the degree of localized
MMP-9 degradation of ECM components. In summary, the present study provides support for the concept of a functional relationship between mast cells and activated T cells involving either soluble mediators secreted from both cell populations or direct cell-cell contact. Herein, we show evidence that direct contact between the two cell types stimulates mast cells to produce and release several granule-associated mediators, cytokines and MMP-9, which possess immunoregulatory and/or immunomodulatory properties. These mast cell activities may support reciprocal activities by other leukocytes, such as cell migration, as well as promote structural and biochemical changes in the ECM microenvironment during T cell-mediated inflammation.
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Footnotes |
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2 D.B. and G.G.V. contributed equally to this work.
3 Address correspondence and reprint requests to Dr. Yoseph A. Mekori, Department of Medicine B, Meir General Hospital, Kfar-Saba, 44281, Israel. E-mail address: ymekori{at}netvision.net.il
4 Abbreviations used in this paper: ECM, extracellular matrix; MMP, matrix metalloproteinase; TIMP, tissue inhibitor of metalloproteinase; SCF, stem cell factor.
Received for publication March 5, 2001. Accepted for publication July 25, 2001.
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and 1,25(OH)2D3 induce on THP-1 cells distinct patterns of cell surface antigen expression, cytokine production and responsiveness to contact with activated T cells. J. Immunol. 149:2040.[Abstract]
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) and MMP-9 (
) release. For the purpose of
comparison between 
), and
cathepsin D (•) were measured as described in Materials and
Methods.
). Data are presented as the percentage of maximal
release, as described in the legend of Fig. 3
