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Volume 272, Number 50, Issue of December 12, 1997
pp. 31764-31769
(Received for publication, June 12, 1997, and in revised form, October 2, 1997)
From the ABSTRACT The IL-1 type II receptor (decoy RII) is a
nonsignaling molecule the only established function of which is to
capture IL-1 and prevent it from interacting with signaling receptor.
The decoy RII is released in a regulated way from the cell surface.
Here, we reported that hydroxamic acid inhibitors of matrix
metalloproteases inhibit different pathways of decoy RII release,
including the following: (a) the slow (18 h) gene
expression-dependent release from monocytes and
polymorphonuclear cells exposed to dexamethasone; (b) rapid
release (minutes) from myelomonocytic cells exposed to tumor necrosis
factor, chemoattractants, or phorbol myristate acetate; (c)
phorbol myristate acetate-induced release from decoy RII-transfected
fibroblasts and B cells. Inhibition of release was associated with
increased surface expression of decoy RII. Inhibitors of other protease
classes did not substantially affect release. However, serine protease
inhibitors increased the molecular mass of the decoy RII released from
polymorphonuclear cells from 45 to 60 kDa. Thus, irrespective of the
pathway responsible for release and of the cellular context, matrix
metalloproteases, rather than differential splicing, play a key role in
production of soluble decoy RII.
INTRODUCTION IL-11 is the name of two
polypeptide mediators (IL-1 Two receptors for IL-1, termed type I and type II (RI and RII,
respectively), usually coexpressed in different cell types, have been
identified and cloned (6-8). An accessory protein, which increases the
binding affinity of RI for IL-1 The decoy RII is released in vitro and is found in
biological fluids in a variety of pathophysiological condition
(14-17). Two main pathways of regulation of decoy RII release have
been identified. Anti-inflammatory signals (e.g.
glucocorticoid hormones) augment decoy RII gene expression and
eventually release in myelomonocytic cells (18, 19). Over a period of
18 h, dexamethasone increases the number of monocyte surface RII
from 171 to 3742 receptors/cell, and about 150 × 106
molecules are released in the culture medium over the same period of
time by 20 × 106 monocytes (19). A second pathway of
regulation of the decoy RII involves the rapid (5 min) shedding from
the cell surface. This rapid pathway of release is activated by
chemoattractants, reactive oxygen intermediates, phorbol myristate
acetate (PMA), and TNF (20-22). The present study was designed to
assess whether proteolytic enzymes are involved in the gene
expression-dependent and -independent pathways of decoy
RII. The identification of an mRNA transcript encoding a released
version of decoy RII (23) raised the issue of the relative contribution
of proteolytic shedding versus differential splicing in
production of soluble decoy RII. The results presented here show that
metalloprotease inhibitors block all pathways of soluble decoy RII
production and, by implication, that proteolytic shedding is a dominant
mechanism for generation of soluble decoy RII.
EXPERIMENTAL PROCEDURES Human PMNs were separated from the peripheral blood
of human healthy donors by Percoll gradient centrifugation (24).
Briefly, whole blood was fractionated by Ficoll gradient centrifugation (Seromed-Biochem KG, Berlin, Germany), and PMNs, collected from the
pellet, were layered on top of 62% Percoll (Pharmacia & Upjohn, Uppsala, Sweden) after a centrifugation at 1500 rpm for 20 min at room
temperature. PMN pellets ( Human fibrosarcoma cells (line 8387) were transfected with pCEP4 The 1H7 cell line is a subline of Epstein-Barr virus-positive Burkitt
lymphoma line Raji obtained by limiting dilution and selection for high
expression of decoy RII (26).
The metalloprotease inhibitor BB-94 was from
British Biotechnology (Oxford, United Kingdom); L,680833 (a specific
elastase inhibitor) and CT1418 and Ro31,9790 (two metalloprotease
inhibitors) were from Pharmacia and Upjohn (Nerviano, Italy); PMA and
fMLP were from Sigma; The assay was conducted as described (20).
Briefly, 12 × 106 cells were incubated with or
without the indicated stimuli in serum-free medium at 37 °C in 5%
CO2 for 20 min in polypropilene 50-ml conical tubes
(Falcon-Beckton Dickinson Labware, Lincoln Park, NJ). Cells were then
washed with binding buffer (RPMI 1640 medium, 0.2% bovine serum
albumin, pH 7.4), and 4 × 106 cells were incubated
with 800 pM 125I-labeled IL-1 30 × 106 cells
were incubated with protease inhibitors and stimuli in 1 ml of RPMI
1640 medium at 37 °C for 20 min. Medium was recovered and
concentrated 10 times by membrane filtration (Amicon, Beverly, MA;
cut-off, 10,000). 100 µl were added to 1 nM
125I-labeled IL-1 The supernatants from 5 × 106 83C7 cells were collected, concentrated, subjected to
10% SDS-PAGE, and then blotted onto a nitrocellulose filter
(Stratagene, La Jolla, CA). Incubation with primary and secondary
antibodies was carried out according to standard protocols. A mouse mAb
against decoy RII (clone
8/5)2 was used as primary
antibody. Immunoreactive protein bands were revealed by a
chemiluminescence-based procedure (ECL Detection, Amersham
International, Little Chalfont, UK) according to the manufacturer's
instructions.
The chemotactic response of human
circulating PMNs to fMLP was tested as described (27, 28). Briefly,
25 ± 1 µl of fMLP, diluted in 1% FCS-RPMI 1640 medium, were
seeded in the lower compartment of the chemotaxis chamber, and 50 µl
of cell suspension (1.5 × 106/ml) were seeded in the
upper compartment. The two compartments were separated by a 5-µm pore
size polyvinylpirrolidone-free polycarbonate filter (Nucleopore Corp.,
Pleasant, CA). Chambers were incubated at 37 °C in air with 5%
CO2 for 90 min. At the end of the incubation, filters were
removed, fixed, and stained with Diff-Quik (Harleco, Gibbstown, NJ),
and five high power oil immersion fields were counted.
Cells were exposed for 24 h to
various stimuli or to medium alone. The supernatants were collected and
tested for MCP-1 production by a recently developed sandwich
enzyme-linked immunosorbent assay with a sensitivity limit of 30 pg/ml
(29).
Statistical significance was assessed
by the Tukey test.
RESULTS In a
preliminary series of assays, we examined whether the protease
inhibitors used in this study affected cellular functions unrelated to
proteolytic activity. BB-94, the matrix metalloprotease inhibitor used
most extensively in the present study, and
[View Larger Version of this Image (32K GIF file)]
IL-1 decoy RII is expressed on PMNs as integral
transmembrane protein, and it is released very rapidly in the
supernatant after treatment of the cells with TNF, chemoattractants,
and PMA. To ask what type(s) of enzyme is involved in the cleavage of
this molecule, the effects of various protease inhibitors on the
release of decoy RII was tested. As shown in Fig.
2A, TNF induced a rapid and
drastic decrease of IL-1 binding to PMNs (17 ± 7% of binding compared with 100% of control binding). The metalloprotease inhibitor BB-94, an hydroxamic acid-based inhibitor of metalloproteases, strongly
inhibited the TNF action (84 ± 4%). Two other specific metalloprotease inhibitors (CT1418 and Ro31,9790) blocked the action of
TNF, although less strongly than BB-94 (67 ± 19% for CT1418 and
59 ± 2% for Ro31,9790). Inhibitors of other classes of
proteases, such as serine proteases (
[View Larger Version of this Image (40K GIF file)]
[View Larger Version of this Image (43K GIF file)]
Monocytes also express high quantities of surface decoy
RII (19). TNF induces rapid (10 min) release of IL-1 decoy RII (22). When we tested the effects of different protease inhibitors (Fig. 4A), we found that only
metalloprotease inhibitors, such as BB-94, CT1418, and Ro31,9790,
inhibited the action of TNF. In particular, BB-94 restored the IL-1
binding capacity of TNF-treated cells to 93 ± 12% of control,
compared with 26 ± 10% of cells treated with TNF alone; CT1418
and Ro31,9790 also caused a significant inhibition of TNF action
(92 ± 8% for CT 1418 and 81 ± 9% for Ro 31,9790). Other
protease inhibitors, such as L,680833 and E-64, had no effect on IL-1
decoy RII release. As shown in Fig. 4B, TNF-stimulated
monocytes released a 60-kDa soluble IL-1 decoy RII, and BB-94 inhibited
the shedding of soluble receptor.
[View Larger Version of this Image (39K GIF file)]
In agreement with previous
reports (18, 19), dexamethasone augmented the surface expression of the
decoy RII (reflected by increased specific binding) in both monocytes
and PMNs (Fig. 5, A and
C). Concomitantly, dexamethasone augmented the release of
soluble versions of decoy RII of 45 and 60 kDa for PMNs and monocytes,
respectively (Fig. 5, B and D). BB-94 blocked
release of the decoy RII from dexamethasone-treated myelomonocytic
cells and concomitantly augmented binding on the cell surface.
[View Larger Version of this Image (50K GIF file)]
The results discussed so
far were obtained with myelomonocytic cells. It was important to assess
whether metalloproteases play a central role in decoy RII release in a
different cellular context as well. We therefore examined the effect of
metalloprotease inhibitors on decoy RII release from B cells (cell line
1H7, a subline of an Epstein-Barr virus-positive Burkitt lymphoma line Raji, selected for high expression of IL-1 decoy RII (26)) and transfected fibroblasts (transfected cell line 8387). Both of these
cell lines released large amounts of soluble decoy RII spontaneously and after stimulation with PMA. As shown in Figs.
6B, 6C, and 7B, BB-94 inhibited the
spontaneous and PMA-induced release of the decoy RII in transfected
fibroblast cells and 1H7 cells. Concomitantly, BB-94 blocked the
PMA-induced reduction of IL-1 binding in these nonmyeloid cellular
contexts (Figs. 6A and 7A). The capacity of BB-94
to block decoy RII release from transfected fibroblasts was observed by
both cross-linking and Western analysis.
[View Larger Version of this Image (33K GIF file)]
[View Larger Version of this Image (36K GIF file)]
DISCUSSION The results presented here show that one or more enzyme of the
metalloprotease class play a key role in the production of soluble
versions of the decoy RII. Different inhibitors of metalloproteases inhibited release of decoy RII, whereas agents which act on serine proteases or cysteine proteases had little or no effect.
Metalloprotease inhibitors did not affect various functions of the
cells used in the present study, including PMN chemotaxis,
lipopolysaccharide-induced cytokine production in monocytes, and
PMA-activated cytokine production in transfected fibroblasts. It is
therefore unlikely that the capacity of different metalloprotease
inhibitors to block decoy RII release is related to effects other than
target enzyme inhibition.
The decoy RII is present in biological fluids, with augmented levels in
inflammatory conditions (16, 17) and in culture supernatants (18, 19).
Previous studies had identified two general pathways through which
environmental signals activate release of the decoy RII in
myelomonocytic cells. Several molecules with anti-inflammatory activity
(glucocorticoids, IL-4, and IL-13) augmented gene expression and
subsequently surface levels of decoy RII: an increased surface
expression is associated with increased release (18, 19). A second
pathway is gene expression- and protein synthesis-independent: decoy
RII release is activated in about 5 min by chemoattractants, reactive
oxygen intermediates, and TNF (20-22). In the present study,
metalloprotease inhibitors blocked decoy RII release irrespective of
the pathway of induction and the cellular context.
The soluble decoy RII present in biological fluids was classically
described as a 45-kDa molecule (16-18). In vitro, we have described two forms of released decoy RII, a 45-kDa molecule released from PMNs (18, 20, 22) and a 60-kDa form released from monocytes and
transfected fibroblasts (19, 25). Intriguingly, given the central role
of monocytes in inflammation, the 60-kDa version of decoy RII had not
previously been described in biological fluids. The results reported
here may provide a reasonable explanation for this apparent
discrepancy. Serine protease inhibitors had little or no effect on
decoy RII release. However, serine protease inhibitors modified the
size of decoy RII released from PMNs. In the presence of the
inhibitors, PMNs released a 60-kDa, rather than 45-kDa, form of the
decoy RII. Thus one could speculate that all cell types release
primarily a 60 kDa version of the decoy RII via metalloproteases, and
then extracellular serine proteases produced by PMNs and possibly other
cellular elements process it to the 45-kDa form found in
vivo. The extracellular domains of different membrane proteins,
including pro-TNF Soluble receptors of different cytokines fulfill different functions
(43, 44). For instance, soluble IL-6 receptor allows interaction of
IL-6 with the signal-transducing gp130 chain (45, 46), whereas soluble
TNF receptor usually blocks TNF (47) and is currently undergoing
clinical evaluation (48). The released IL-1 decoy RII does not
facilitate signal transduction (10) and presumably acts as a systemic
buffering system for IL-1 (3, 13).
A splice variant of the decoy RII mRNA, encoding a molecule without
a transmembrane segment, has been identified (23). The results reported
here show that metalloprotease inhibitors block release and augment
surface expression of the decoy RII, irrespective of the inducing agent
and the cellular context. Therefore, these observations suggest that
proteolytic shedding, rather than differential RNA splicing, is the
dominant pathway for generation of soluble decoy RII.
FOOTNOTES REFERENCES
Role of Metalloproteases in the Release of the IL-1 type II
Decoy Receptor*
§,,,
,§§
Department of Immunology and Cell Biology,
Istituto di Ricerche Farmacologiche "Mario Negri," Via Eritrea 62, 20157 Milano, Italy; ¶ British Biotech, Cowley, Oxford OX4 5LY,
United Kingdom; 
Research Center Dompé Spa, I-67100
L'Aquila, Italy; ** Department of Immunology, Pharmacia & Upjohn
Research Center, 20014 Nerviano, Italy; and
Department of Biotechnology, Section of
General Pathology, University of Brescia, 25100 Brescia, Italy
and IL-1
) that are among the most
potent and multifunctional cell activators described in immunology and
cell biology literature. The spectrum of action of IL-1 encompasses
cells of hematopoietic origin, from immature precursors to
differentiated leukocytes, vessel wall elements, and cells of
mesenchymal, nervous, and epithelial origin (1, 2). The production and
action of IL-1 are regulated by multiple control pathways, some of
which unique to this cytokine. This complexity and uniqueness is best
represented by the term "IL-1 system" (3). The IL-1 system consists
of two agonists, IL-1 and IL-1, a specific activation system
(IL-1-converting enzyme), a receptor antagonist produced in different
isoforms (4, 5), and two high affinity surface binding molecules (3).
, has recently been identified (9).
IL-1 signaling activity appears to be mediated exclusively via the RI
and the accessory protein (9, 10-12), whereas the IL-1RII has no
signaling property and acts in myelomonocytic cells as a decoy for IL-1
(decoy RII), inhibiting its activity by preventing IL-1 from binding to
the signaling RI (3, 13).
98% pure as assessed by morphology) were
resuspended at 107 cells/ml in RPMI 1640 medium
(Seromed-Biochem KG) with 2 mM glutamine (Seromed-Biochem
KG). Human monocytes were separated from the peripheral blood of human
healthy donors by Percoll gradient centrifugation (24). Briefly, whole
blood was fractionated by Ficoll-Hypaque gradient centrifugation
(Seromed-Biochem KG), and mononuclear cells, collected from the ring,
were layered on top of 46% Percoll (Pharmacia & Upjohn) after a
centrifugation at 2000 rpm for 30 min at room temperature. Monocytes
(98% pure as assessed by morphology) were resuspended and incubated
(5 × 106 cells/ml) for 40 h in RPMI 1640 medium
with 2 mM glutamine and 30% autologous serum on
hydrophobic plates (Petriperm Hydrofob, Heraeus, Osterode,
Germany).
episomal expression vector containing the IL-1RII full-length cDNA
(25). 83C7 refers to cells containing the IL-1Rll. 83C7 cells have
2 × 103 (clone A) or 4 × 104 (clone
B) IL-1 binding sites/cell. The receptor released by 83C7 cells is
similar in size to that of monocytes (see below) and was identified as
the decoy RII by cross-linking, antibody blocking, and Western analysis
(19).
1-AT and E-64 were from
Calbiochem; TNF was from BASF Knoll (Ludwigshaven, Germany). Media
and reagents contained less than 0.125 units/ml of endotoxin as checked
by Limulus Amebocyte Lysate assay (BioWhittaker, Walkerville, MD).
(specific
activity, 180 µCi/µg; NEN Life Science Products, Bad Homburg,
Germany) in the presence or absence of a 100-fold molar excess of cold
cytokine in 50 µl of binding buffer at 4 °C for 4 h in
polystyrene 96-well round-bottomed microplates (Falcon) on a shaking
platform. Preliminary experiments showed that binding reached a plateau
at 4 h under these conditions. To separate bound from free
125I-labeled IL-1, cells were resuspended, transferred
to Eppendorf tubes, washed in binding buffer, resuspended in 70 µl of
binding buffer, layered on the top of a 200-µl cushion of 20%
sucrose (Merck), 1% bovine serum albumin in 400-µl polypropylene
tubes (Beckman Instruments), and centrifuged at 10,000 rpm for 30 s at room temperature. The cellular pellets were counted in a
-counter.
, and incubated at 4 °C for 4 h. After addition of 1 mM disuccinimidyl suberate (DSS)
(Pierce) at 4 °C for 30 min, samples were analyzed by 8% SDS-PAGE
under reducing conditions, and dried gels were exposed to
autoradiography for 1-3 days. Nonspecific binding was determined by
adding a 200-fold molar excess of unlabeled IL-1, 10 µg/ml M1
(blocking mAb anti-IL-1 RI), or M22 (blocking mAb anti-IL-1 RII)
(kindly provided by Dr. J. E. Sims (Immunex)) (data not
shown).
1-AT did not
affect PMN chemotaxis, lipopolysaccharide-induced MCP-1 production in
monocytes, or PMA-activated MCP-1 production in 83C7 cells (Fig.
1).
Fig. 1.
Effect of BB-94 on PMN and monocyte
functions. A, effect of BB-94 and 1-AT on
fMLP-induced PMN chemotaxis: PMNs were incubated with
1-AT (100 µg/ml) or BB-94 (1 µg/ml), and then fMLP
chemotactic response was tested (see "Experimental Procedures"). Data are expressed as migrated cells (means with range of two different
donors). B, effect of BB-94 on MCP-1 production induced by
TNF and lipopolysaccharide in monocytes: monocytes were incubated with
medium alone or with lipopolysaccharide (10 ng/ml) or TNF (50 ng/ml)
with or without BB-94 (1 µg/ml) for 24 h at 37 °C in RPMI
1640 medium with 1% FCS, and then MCP-1 production was tested (see
"Experimental Procedures"). Data are expressed as ng/ml of MCP-1
released by 2 × 105 monocytes (mean with range of two
different donors). C, effect of BB-94 on MCP-1 production
induced by PMA in IL-1 decoy RII-transfected cell line 8387: cells were
incubated with medium alone or PMA (50 ng/ml) with or without BB-94 (1 µg/ml) for 24 h at 37 °C in RPMI 1640 medium with 1% FCS,
and then MCP-1 production was tested (see "Experimental
Procedures"). Data are expressed as ng/ml of MCP-1 released by 4 × 104 cells (mean with range of two different
experiments).
1-AT and L,680833)
and cystein proteases (E-64), did not affect TNF-induced release of IL-1 decoy RII. The increase in the amount of IL-1 binding at the level
of cell-associated receptor was reflected by a concomitant decrease of
the soluble receptor in the supernatant as shown in Fig. 2B.
More precisely, TNF increased the level of a soluble IL-1 binding
molecule of 45 kDa, as expected. BB-94 strongly inhibited TNF action;
surprisingly, treatment of PMNs with 1-AT, a serine protease inhibitor, did not cause inhibition of TNF action but shifted
the size of the released decoy RII from 45 to 60 kDa, the same
molecular mass observed in monocytes and IL-1 decoy RII-transfected cells (19, 25). L,680833, a specific elastase inhibitor, also caused
the shift in the released decoy RII from 45 to 60 kDa (data not shown).
Chemoattractant molecules, such as fMLP and PMA, induced rapid release
of decoy RII in PMNs (20, 22). We tested the effect of BB-94 on release
of IL-1 decoy RII induced by these two stimuli. As shown in Fig.
3, fMLP and PMA induced rapid release of
decoy RII as reflected by decrease in binding to cells (32 ± 7%
and 48 ± 4% for fMLP and PMA, respectively). As expected, BB-94
inhibited the release induced by fMLP and PMA (81 ± 11% and
83 ± 2% of control values, respectively).
Fig. 2.
Effect of protease inhibitors on TNF-induced
rapid release of the IL-1 decoy RII in PMNs. A, effect on
surface binding: 10 × 106 PMNs were incubated with
BB-94 (1 µg/ml), CT1418 (10 µM), Ro 31,9790 (10 µM), E-64 (10 µM), L,680833 (2 µM), or 1-AT (100 µg/ml) with or without
TNF (50 ng/ml) for 20 min at 37 °C in serum-free RPMI 1640 medium
and then examined for IL-1 binding. Data are shown as percentage of
control of specific binding of IL-1 (mean ± S.D. of three
different donors). B, effect on release in the supernatant:
30 × 106 PMNs were cultured with or without TNF (50 ng/ml) in the presence of absence of BB-94 (1 µg/ml) and
1-AT (100 µg/ml) for 20 min at 37 °C in serum-free
RPMI 1640 medium. Supernatants were then recovered, concentrated, mixed
with 125I-labeled IL-1, cross-linked with DSS, and then
analyzed by SDS-PAGE. Nonspecific binding was determined by adding a
200-fold molar excess of unlabeled IL-1, 10 µg/ml M1 (blocking mAb
anti-IL-1 RI), or 10 µg/ml M22 (blocking mAb anti-IL-1 RII) (kindly
provided by Dr. J. E. Sims (Immunex)) (data not shown).
Fig. 3.
Effect of protease inhibitors on fMLP- and
PMA-induced release of the IL-1 decoy RII in PMNs. 10 × 106 PMNs were incubated with TNF (50 ng/ml), fMLP
(10
7 M), or PMA (50 ng/ml) for 20 min at
37 °C in serum-free RPMI 1640 medium with or without BB-94 (1 µg/ml) and then examined for IL-1 binding. Data are shown as
percentage of control of specific binding of IL-1 (mean ± S.D. of
three different donors).
Fig. 4.
Effect of protease inhibitors on TNF-induced
release of the IL-1 decoy RII in monocytes. A, effect on
surface binding: 10 × 106 monocytes were incubated
with BB-94 (1 µg/ml), CT1418 (10 µM), Ro31,9790 (10 µM), E-64 (10 µM), or L,680833 (2 µM), with or without TNF (50 ng/ml) for 20 min at
37 °C in serum-free RPMI 1640 medium and then examined for IL-1
binding. Data are shown as percentage of control of specific binding of
IL-1 (mean ± S.D. of three different donors). B,
effect on release in the supernatant: 30 × 106
monocytes were cultured with or without TNF (50 ng/ml) in the presence
or absence of BB-94 (1 µg/ml) for 20 min at 37 °C in serum-free
RPMI 1640 medium. Supernatants were then recovered, concentrated, mixed
with 125I-labeled IL-1, cross-linked with DSS, and then
analyzed by SDS-PAGE.
Fig. 5.
Effect of protease inhibitors on
dexamethasone-induced release of the IL-1 decoy RII in PMNs and
monocytes. A, effect on PMN surface binding: 10 × 106 PMNs were incubated with or without dexamethasone
(106 M) in the presence or absence of BB-94
(1 µg/ml) for 18 h at 37 °C in RPMI 1640 medium with 10% FCS
and then examined for IL-1 binding. Data are shown as cpm of specific
binding of 125I-labeled IL-1 of a representative
experiment out of three experiments performed. B, effect on
release in the supernatant by PMNs: 30 × 106 PMNs
were cultured with or without dexamethasone (106
M) in the presence or absence of BB-94 (1 µg/ml) for
18 h at 37 °C in serum-free RPMI 1640 medium. Supernatants were
then recovered, concentrated, mixed with 125I-labeled
IL-1, cross-linked with DSS, and then analyzed by SDS-PAGE. C, effect on monocyte surface binding: 10 × 106 monocytes were incubated with or without dexamethasone
(106 M) in the presence or absence of BB-94
(1 µg/ml) for 18 h at 37 °C in RPMI 1640 medium with 10% FCS
and then examined for IL-1 binding. Data are shown as cpm of specific
binding of 125I-labeled IL-1 of a representative
experiment out of three experiments performed. D, effect on
release in the supernatant by monocytes: 30 × 106
monocytes were cultured with or without dexamethasone
(106 M) in the presence or absence of BB-94
(1 µg/ml) for 18 h at 37 °C in serum-free RPMI 1640 medium.
Supernatants were then recovered, concentrated, mixed with
125I-labeled IL-1, cross-linked with DSS, and then
analyzed by SDS-PAGE.
Fig. 6.
Effect of protease inhibitors on PMA-induced
release of the IL-1 decoy RII on IL-1 decoy RII-transfected 8387 cell
line. A, effect on surface binding: 3 × 106 83C7 cells were incubated with 1-AT (100 µg/ml), BB-94 (1 µg/ml), or E-64 (10 µM), with or
without PMA (50 ng/ml) for 20 min at 37 °C in serum-free RPMI 1640 medium and then examined for IL-1 binding. Data are shown as percentage
of control of specific binding of IL-1 (mean with range of two
different experiments). B, effect on release in the
supernatant (cross-linking): 10 × 106 83C7 (clone A;
2 × 103 receptors/cell) cells were cultured with or
without PMA (50 ng/ml) in the presence or absence of BB-94 (1 µg/ml)
for 20 min at 37 °C in serum-free RPMI 1640 medium. Supernatants
were then recovered, concentrated, mixed with 125I-labeled
IL-1, cross-linked with DSS, and then analyzed by SDS-PAGE. C, effect on release in the supernatant (Western blotting):
10 × 106 83C7 (clone B; 4 × 104
receptors/cell) cells were cultured with or without PMA (50 ng/ml) in
the presence or absence of BB-94 (1 µg/ml) for 20 min at 37 °C in
serum-free RPMI 1640 medium. Supernatants were then recovered, concentrated, run on 10% SDS-PAGE, and then analyzed by Western blot
with anti-RII mAb 8/5.
Fig. 7.
Effect of protease inhibitors on PMA-induced
release of the IL-1 decoy RII on 1H7 cell line. A, effect on
surface binding: 3 × 106 1H7 cells were incubated
with 1-AT (100 µg/ml), BB-94 (1 µg/ml), or E-64 (10 µM), with or without PMA (50 ng/ml) for 20 min at 37 °C in serum-free RPMI 1640 medium and then examined for IL-1 binding. Data are shown as percentage of control of specific binding of
IL-1 (mean with range of two different experiments). B,
effect on release in the supernatant: 10 × 106 cell
were cultured with or without PMA (50 ng/ml) in the presence or absence
of BB-94 (1 µg/ml) for 20 min at 37 °C in serum-free RPMI 1640 medium. Supernatants were then recovered, concentrated, mixed with
125I-labeled IL-1, cross-linked with DSS, and then
analyzed by SDS-PAGE.
, p75 and p55 TNF receptor, IL-6-receptor,
Fas-ligand, L-selectin, pro-transforming growth factor , and
thyrotropin receptor are shed in the supernatant in different cell
type, by one or more metalloproteases (30-38). A mutant Chinese
hamster ovary cell line, defective in the shedding of several unrelated
membrane proteins, was recently described, suggesting a common system
for membrane protein shedding (39). On the other hand, no sequence
similarity can be found in the cleavage site of released proteins (39).
Studies based on mutational analysis of the cleavage site of
L-selectin, p55 TNF receptor, and pro-TNF (40-42) showed that the
proteolytic processing of molecules might depend on the secondary
structural characteristics of the cleavage domain and might not require
strict sequence specificity. The observation that the juxtamembrane
region of the decoy RII shows no primary sequence similarity to other
released receptors does not necessarily imply the involvement of
different enzyme systems.
§
S. Orlando is a Banca di Roma fellow.
§§
To whom correspondence should be addressed: Department of
Immunology and Cell Biology, Istituto di Ricerche Farmacologiche "Mario Negri," Via Eritrea 62, 20157 Milano, Italy. Tel.:
39-2-390141; Fax: 39-2-3546277; E-mail:
mantovani{at}irfmn.mnegri.it.
1
The abbreviations used are: IL, interleukin; RI,
IL-1 type I receptor; RII, IL-1 type II receptor; PMN,
polymorphonuclear cell; TNF, tumor necrosis factor; PMA, phorbol
myristate acetate; fMLP, formyl-Met-Leu-Phe; 1-AT,
1-antitrypsin inhibitor; MCP-1, monocyte chemotactic
protein-1; DSS, disuccinimidyl suberate; PAGE, polyacrylamide gel
electrophoresis; mAb, monoclonal antibody; FCS, fetal calf serum.
2
G. Peri, unpublished data.
Volume 272, Number 50,
Issue of December 12, 1997
pp. 31764-31769
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.
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  C. Bellehumeur, J. Blanchet, J.-Y. Fontaine, N. Bourcier, and A. Akoum Interleukin 1 regulates its own receptors in human endometrial cells via distinct mechanisms Hum. Reprod., May 28, 2009; (2009) dep192v1. [Abstract] [Full Text] [PDF]
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 P. Van Lint and C. Libert Chemokine and cytokine processing by matrix metalloproteinases and its effect on leukocyte migration and inflammation J. Leukoc. Biol., December 1, 2007; 82(6): 1375 - 1381. [Abstract] [Full Text] [PDF]
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 C. Herrmann-Lavoie, C. V. Rao, and A. Akoum Chorionic Gonadotropin Down-Regulates the Expression of the Decoy Inhibitory Interleukin 1 Receptor Type II in Human Endometrial Epithelial Cells Endocrinology, November 1, 2007; 148(11): 5377 - 5384. [Abstract] [Full Text] [PDF]
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 S Guay and A Akoum Stable inhibition of interleukin 1 receptor type II in Ishikawa cells augments secretion of matrix metalloproteinases: possible role in endometriosis pathophysiology Reproduction, September 1, 2007; 134(3): 525 - 534. [Abstract] [Full Text] [PDF]
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A. Akoum, C. Lawson, C. Herrmann-Lavoie, and R. Maheux Imbalance in the expression of the activating type I and the inhibitory type II interleukin 1 receptors in endometriosis Hum. Reprod., May 1, 2007; 22(5): 1464 - 1473. [Abstract] [Full Text] [PDF]
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 P.-H. Kuhn, E. Marjaux, A. Imhof, B. De Strooper, C. Haass, and S. F. Lichtenthaler Regulated Intramembrane Proteolysis of the Interleukin-1 Receptor II by {alpha}-, beta-, and {gamma}-Secretase J. Biol. Chem., April 20, 2007; 282(16): 11982 - 11995. [Abstract] [Full Text] [PDF]
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 E. Simeoni, J. Dudler, S. Fleury, J. Li, M. Pagnotta, M. Pascual, L. K. von Segesser, and G. Vassalli Gene transfer of a soluble IL-1 type 2 receptor-Ig fusion protein improves cardiac allograft survival in rats Eur. J. Cardiothorac. Surg., February 1, 2007; 31(2): 222 - 228. [Abstract] [Full Text] [PDF]
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 G. Verbruggen Chondroprotective drugs in degenerative joint diseases Rheumatology, February 1, 2006; 45(2): 129 - 138. [Abstract] [Full Text] [PDF]
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C. Bellehumeur, T. Collette, R. Maheux, J. Mailloux, M. Villeneuve, and A. Akoum Increased soluble interleukin-1 receptor type II proteolysis in the endometrium of women with endometriosis Hum. Reprod., May 1, 2005; 20(5): 1177 - 1184. [Abstract] [Full Text] [PDF]
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 L. E. Jensen and A. S. Whitehead The 3' Untranslated Region of the Membrane-Bound IL-1R Accessory Protein mRNA Confers Tissue-Specific Destabilization J. Immunol., November 15, 2004; 173(10): 6248 - 6258. [Abstract] [Full Text] [PDF]
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S. J. Levine Mechanisms of Soluble Cytokine Receptor Generation J. Immunol., November 1, 2004; 173(9): 5343 - 5348. [Abstract] [Full Text] [PDF]
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 Y. Yang, W. Bin, M.O. Aksoy, and S.G. Kelsen Regulation of interleukin-1{beta} and interleukin-1{beta} inhibitor release by human airway epithelial cells Eur. Respir. J., September 1, 2004; 24(3): 360 - 366. [Abstract] [Full Text] [PDF]
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X. Cui, F. N. Rouhani, F. Hawari, and S. J. Levine Shedding of the Type II IL-1 Decoy Receptor Requires a Multifunctional Aminopeptidase, Aminopeptidase Regulator of TNF Receptor Type 1 Shedding J. Immunol., December 15, 2003; 171(12): 6814 - 6819. [Abstract] [Full Text] [PDF]
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E. Bourke, A. Cassetti, A. Villa, E. Fadlon, F. Colotta, and A. Mantovani IL-1{beta} Scavenging by the Type II IL-1 Decoy Receptor in Human Neutrophils J. Immunol., June 15, 2003; 170(12): 5999 - 6005. [Abstract] [Full Text] [PDF]
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B. Muller, G. Peri, A. Doni, A. P. Perruchoud, R. Landmann, F. Pasqualini, and A. Mantovani High circulating levels of the IL-1 type II decoy receptor in critically ill patients with sepsis: association of high decoy receptor levels with glucocorticoid administration J. Leukoc. Biol., October 1, 2002; 72(4): 643 - 649. [Abstract] [Full Text] [PDF]
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 A. Kharfi, A. Boucher, and A. Akoum Abnormal Interleukin-1 Receptor Type II Gene Expression in the Endometrium of Women with Endometriosis Biol Reprod, February 1, 2002; 66(2): 401 - 406. [Abstract] [Full Text] [PDF]
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 J. G. Conway, R. C. Andrews, B. Beaudet, D. M. Bickett, V. Boncek, T. A. Brodie, R. L. Clark, R. C. Crumrine, M. A. Leenitzer, D. L. McDougald, et al. Inhibition of Tumor Necrosis Factor-alpha (TNF-alpha ) Production and Arthritis in the Rat by GW3333, a Dual Inhibitor of TNF-alpha -Converting Enzyme and Matrix Metalloproteinases J. Pharmacol. Exp. Ther., September 1, 2001; 298(3): 900 - 908. [Abstract] [Full Text]
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 M A Dentener, E C Creutzberg, A M W J Schols, A Mantovani, C van`t Veer, W A Buurman, and E F M Wouters Systemic anti-inflammatory mediators in COPD: increase in soluble interleukin 1 receptor II during treatment of exacerbations Thorax, September 1, 2001; 56(9): 721 - 726. [Abstract] [Full Text] [PDF]
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 M. Lindsey, K. Wedin, M. D. Brown, C. Keller, A. J. Evans, J. Smolen, A. R. Burns, R. D. Rossen, L. Michael, and M. Entman Matrix-Dependent Mechanism of Neutrophil-Mediated Release and Activation of Matrix Metalloproteinase 9 in Myocardial Ischemia/Reperfusion Circulation, May 1, 2001; 103(17): 2181 - 2187. [Abstract] [Full Text] [PDF]
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 C. L. Cubitt, R. N. Lausch, and J. E. Oakes Synthesis of Type II Interleukin-1 Receptors by Human Corneal Epithelial Cells but not by Keratocytes Invest. Ophthalmol. Vis. Sci., March 1, 2001; 42(3): 701 - 704. [Abstract] [Full Text]
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D. Neumann, C. Kollewe, M. U. Martin, and D. Boraschi The Membrane Form of the Type II IL-1 Receptor Accounts for Inhibitory Function J. Immunol., September 15, 2000; 165(6): 3350 - 3357. [Abstract] [Full Text] [PDF]
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 K. R. Coulter, M. D. Wewers, M. P. Lowe, and D. L. Knoell Extracellular Regulation of Interleukin (IL)-1beta through Lung Epithelial Cells and Defective IL-1 Type II Receptor Expression Am. J. Respir. Cell Mol. Biol., May 1, 1999; 20(5): 964 - 975. [Abstract] [Full Text]
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 A. Price, Q. Shi, D. Morris, M. E. Wilcox, P. M. A. Brasher, N. B. Rewcastle, D. Shalinsky, H. Zou, K. Appelt, R. N. Johnston, et al. Marked Inhibition of Tumor Growth in a Malignant Glioma Tumor Model by a Novel Synthetic Matrix MetalloproteinaseInhibitor AG3340 Clin. Cancer Res., April 1, 1999; 5(4): 845 - 854. [Abstract] [Full Text] [PDF]
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G. Penton-Rol, S. Orlando, N. Polentarutti, S. Bernasconi, M. Muzio, M. Introna, and A. Mantovani Bacterial Lipopolysaccharide Causes Rapid Shedding, Followed by Inhibition of mRNA Expression, of the IL-1 Type II Receptor, with Concomitant Up-Regulation of the Type I Receptor and Induction of Incompletely Spliced Transcripts J. Immunol., March 1, 1999; 162(5): 2931 - 2938. [Abstract] [Full Text] [PDF]
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R. Rahimpour, G. Mitchell, M. H. Khandaker, C. Kong, B. Singh, L. Xu, A. Ochi, R. D. Feldman, J. G. Pickering, B. M. Gill, et al. Bacterial Superantigens Induce Down-Modulation of CC Chemokine Responsiveness in Human Monocytes Via an Alternative Chemokine Ligand-Independent Mechanism J. Immunol., February 15, 1999; 162(4): 2299 - 2307. [Abstract] [Full Text] [PDF]
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