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*
Department of Physiology and Pathology, University of Trieste, Trieste, Italy;
IRCCS Burlo Garofalo, Trieste, Italy; and
Institute of Histology and Embryology, University of Padova, Padova, Italy
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Two distinct species of surface receptors, one of 55 kDa (TNF-R55) and one of 75 kDa (TNF-R75), mediate biological responses to TNF (10). It is well established that the surface expression of both receptors decreases considerably when PMN are exposed to TNF and various agents including FMLP, the tumor promoter PMA, the Ca2+ ionofore A23187, endotoxin, and GM-CSF (11, 12, 13). The decrease in receptor expression occurs via a process known as shedding, whereby the extracellular portion of the receptors is proteolytically cleaved and released in a soluble form that maintains the ability to bind TNF (11, 14). With TNF, receptor down-modulation has been shown to occur via internalization of TNF-R55 and shedding of TNF-R75 (14). Receptor down-modulation may be a mechanism for PMN self-protection from excessive TNF-induced activation in the circulation, during their emigration into the tissues or at inflammatory sites. Indeed, it has been shown that preincubation of PMN with TNF or other stimuli results in PMN deactivation toward subsequent stimulation by TNF, and that this deactivation correlates to a reduction in TNF surface receptors (12).
A TNF-specific deactivation of granulocytes that, similar to in vitro observations, correlated with a decrease in receptor expression, has also been observed in vivo in subjects infused with TNF, in PMN from patients exposed to activated complement during hemodialysis (15), and PMN isolated from aseptic exudates (16). The released receptors, in turn, depending on their concentration relative to that of TNF, have been proposed either to compete with their membrane-bound counterparts and limit TNF bioavailability (17) or to stabilize the ligand thus enhancing TNF signaling (18).
Because PMN rapidly and extensively release their TNF receptors after adherence or exposure to various agonists (11, 12, 13), it has been suggested that these cells might be a significant source of increased circulating TNF receptors observed in physiologic and pathologic conditions such as exercise (19), experimental endotoxemia (20), a clinical model of postoperative sepsis (21), and high-dose TNF in isolated limb perfusion (22). In light of these observations, the comprehension of the mechanisms that regulate shedding in PMN might provide the basis for experimental approaches aimed at modulating systemic responses to TNF.
We undertook this study to characterize the TNF-induced release of TNF
receptors and to determine the nature of the proteinase(s) involved in
the process. Our data show that TNF induces the release of both
receptors, that such a release is mediated by TNF-R55, and that a
metalloproteinase is involved in the process. The finding that the
releasing activity is cell associated and unaffected by inhibitors of
matrix metalloproteinases (MMP) suggests that it may belong to the ADAM
(a disintegrin and metalloproteinase) family of membrane-bound
metalloproteinases. On the basis of the available evidence, the
possibility that TACE (TNF-
converting enzyme) might be the TNF
receptors "sheddase" of stimulated PMN is discussed.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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BSA fraction V, FMLP, and streptavidin-R-PE conjugate were from Sigma (St. Louis, MO); Percoll was obtained from Pharmacia (Uppsala, Sweden). Immunoassays for human soluble TNF-R55 and TNF-R75 were performed using ELISA kits obtained from R&D Systems (Minneapolis, MN). All solutions were made in endotoxin-free water for clinical use.
Proteinases inhibitors
1-Antitrypsin, chymostatin,
1,10-phenanthroline, phosphoramidon, leupeptin, thiorphan,
N--p-tosyl-L-lysine
chloromethyl ketone (TLCK) were obtained from Sigma;
diisopropylfluorophosphate (DFP) was purchased from Acros Organics
(Fair Lawn, NJ);
[4-(N-hydroxyamino)-2R-isobutyl-3S-methylsuccinyl]-L-3-(5,6,7,8-tetrahydro-1-naphthyl)alanine-N-methylamide(KB8301)
(23),
(4-hydroxy-2R-isobutyl-3S-methylsuccinyl)-L-3-(5,6,7,8-tetrahydro-1-naphthyl)alanine-N-methylamide
(KB8845), and
[4-(N-hydroxyamino)-2R-isobutyl-3S-methylsuccinyl]-L-phenylglycine-N-methylamide
(KB7785) (24) were gently provided by Dr. K. Yoshino
(Nippon Organon, Osaka, Japan). TIMP-1 and TIMP-2 were prepared as
described elsewhere (25).
Abs and cytokines
mAb H398 (subclass IgG2a), a TNF-R55-specific and competing mAb (26), was a generous gift of Dr. P. Scheurich (University of Stuttgart, Stuttgart, Germany); mAb utr-1 (subclass IgG1), a TNF-R75-specific and competing mAb (27), was kindly provided by Dr. M. Brockhaus (Hoffmann-LaRoche, Basel, Switzerland); mAb huTACE-M222 (subclass IgG2a), a human TACE-specific mAb (28), was kindly donated by Dr. R. Black (Immunex, Seattle, WA). mAb 2F7 and mAb 16E8 (29, 30), which are rat anti-human ADAM-12 disintegrin-like and cystein-like domains, respectively, and antiserum 119, a rabbit anti-human ADAM-12 disintegrin-like domain polyclonal Ab, were generously provided by Dr. F. Loechel (University of Copenhagen, Copenhagen, Denmark). Affinity isolated biotin-conjugated goat anti-mouse IgG and affinity isolated FITC-labeled goat anti-rat IgG, utilized as secondary Abs in FACS experiments, were obtained from Sigma and Tago (Burlingame CA), respectively. Pure recombinant human TNF, expressed in Escherichia coli, was obtained from Bissendorf Biochemicals (Hannover, Germany). p55TNF, the human TNF mutant that specifically recognizes TNF-R55, and p75TNF, the human TNF mutant that specifically recognizes TNF-R75 (31), were generously donated by Dr. H. Loetscher (Hoffmann-LaRoche).
Neutrophil isolation
Blood was drawn from healthy volunteers and anticoagulated with 4 mM EDTA. Neutrophils were isolated according to a single-step separation procedure (32) with slight modifications, as previously described in detail (33). The resulting cell population contained 96–98% neutrophils, 2–4% eosinophils, and 1–2% mononuclear cells. The cells were separated and suspended in 140 mM NaCl, 5 mM KCl, 5 mM HEPES, 5 mM glucose, and 0.2% BSA, pH 7.4 (HBS--BSA) at room temperature and in the absence of divalent cations to avoid neutrophil aggregation and activation. All experiments were carried out in HBS--BSA supplemented with 1 mM CaCl2 and 1 mM MgCl2 (HBS-BSA).
U937 cells
U937 cells, a human monoblast-like cell line, were cultured in RPMI 1640 medium supplemented with 10% FCS, 2 mM glutamine, and 50 µg/ml gentamicin at 37°C in an atmosphere of 95% air and 5% CO2.
Radioassay of TNF receptor expression
The radioiodination of TNF was performed as previously described (33). After incubation for 15 min in the presence or in the absence of TNF, neutrophils were cooled in ice and centrifuged for 5 min at 200 x g. All subsequent steps were conducted at 4°C to prevent cell activation and recycling and/or shedding of TNF receptors. The cells were then incubated with a glycine-HCl buffer (50 mM glycine and 125 mM NaCl (pH 3.0)) for 1.5 min to remove any TNF bound to the receptors. Preliminary experiments revealed that this procedure removes all cell-associated 125I-labeled TNF (125I-TNF) without affecting cell viability as shown by others (14, 34). To stop the acid treatment, the cells were diluted with HBS-BSA and centrifuged, and the pellets were resuspended in HBS-BSA. Each cell suspension was divided in aliquots and used for receptors measurement. Expression of total TNF receptors was measured after addition of 2.5 ng/ml 125I-TNF (sp. act. 33.0 or 45.0 µCi/µg). For expression of TNF-R55, mAb utr-1 (10 µg/ml) was included in the incubation mixture, in addition to 125I-TNF, to block binding of TNF to TNF-R75. For expression of TNF-R75, mAb H398 (10 µg/ml) was included in the incubation mixture, in addition to 125I-TNF, to block binding of TNF to TNF-R55. Nonspecific binding was determined by addition of a 200-fold excess of cold TNF. After incubation for 2.5 h under agitation on a rocker platform, the unbound TNF was removed by centrifugation (30 s at 13,000 x g, 4°C) and two washings with HBS-BSA. The pellet-containing bottom parts of the 1.5-ml microfuge tubes used in the experiments were then cut off and counted in a gamma counter.
Assay of TNF receptor releasing activity in PMN supernatants
PMN (107/ml) were incubated without and with FMLP (5 x 10-8 M) for 20 min at 37°C under constant agitation. After cooling on ice, the cells were centrifuged (13,000 x g for 3 min) and the supernatants collected. In parallel experiments, U937 cells were collected, centrifuged at 250 x g for 7 min, and suspended in HBS-BSA. Aliquots of supernatants corresponding to 7.5 x 106 cells, from resting and FMLP-treated PMN were then incubated with 106 U937 cells for 20 min at 37°C. After centrifugation, expression of TNF receptors on U937 cells was measured by flow cytometry.
Immunofluorescence flow cytometry
U937 cells or PMN after the different treatments were cooled at 4°C, divided in aliquots, and incubated for 45 min with the Abs of various specificities: mAb H398 (2 µg/ml) for TNF-R55, mAb utr-1 (2 µg/ml) for TNF-R75, mAb M222 (3 µg/ml) for TACE, and mAbs 2F7 and 16E8 (5 µg/ml) for ADAM-12. After two washes with ice-cold PBS, the cells were incubated for 30 min with biotinylated goat anti-mouse IgG and, after two additional washes, were incubated for another 20 min with a streptavidin-R-PE conjugate. For mAbs 2F7 and 16E8, a FITC-conjugated goat anti-rat IgG secondary Ab was used. After washing, the cells were suspended in PBS containing 0.75% formaldehyde and analyzed by flow cytometry (FACScan, Becton Dickinson, San Jose, CA).
Measurement of solubilized TNF receptors
A commercially available ELISA kit (R&D Systems) was used to measure the TNF receptors released in the supernatants obtained from PMN after the various treatments. In this assay an immobilized anti-TNF-R55 or anti-TNF-R75 mAb is used to capture the specific receptor, which is then detected by a peroxidase-conjugated polyclonal Ab. According to the manufacturer, in this immunoassay, TNF does not show any significant cross-reactivity and exhibits only a low level interference (10% decrease in the observed value using TNF at 5 ng/ml).
In all experiments the spontaneously solubilized receptors before the start of each experiment (t0) were subtracted from the final results.
Data analysis
ID50 values were determined by fitting the data to a sigmoidal curve using GraphPad Prism 3.0 for Windows 98 (GraphPad Software, San Diego, CA). Student’s t test on paired data was used to calculate statistical significance.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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PMN were incubated in the absence and in the presence of TNF, and
TNF receptor expression and shedding were measured. Fig. 1
A shows that TNF causes
down-modulation of the expression of its receptors, and that this is
accompanied by their release in the incubation medium (Fig. 1B). In quantitative terms, the release of TNF-R75 is higher
than that of TNF-R55. Also, resting release of TNF-R75 is more marked
than the corresponding release of TNF-R55. The total amount of
receptors measured in Triton X-100 extracts of PMN was 69.0 ± 3.4
and 101.9 ± 17.3 (pg/106 PMN ± SD,
n = 3) for TNF-R55 and TNF-R75, respectively. Thus,
considering that the extra amount of shed receptors in the presence of
TNF (TNF minus resting) is 16.1 ± 6.7 for TNF-R55 and 45.0
± 16.4 for TNF-R75 (Fig. 1), it turns out that TNF causes the release
of about 23% of TNF-R55 and 44% of TNF-R75. Fig. 2 shows that TNF-induced release of both
receptors becomes apparent after a lag time of 5 min from the addition
of the cytokine, increases rapidly afterward, and reaches its maximum
after 15 min of incubation. In contrast, release from resting PMN
linearly increases with time, at least up to the 60 min of incubation
of our experiments.
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Initially, we tested the effect of receptor-specific reagents on
receptor shedding. Fig. 3 shows
that, after 15 min of incubation, p55TNF, a TNF-R55 specific
TNF mutant, induces a release of both receptors, which is comparable to
that induced by TNF. In contrast, p75TNF, a TNF-R75-specific mutant,
does not exert any effect, even after 30 min of incubation (data not
shown). The possibility that shedding could have occurred after TNF-R75
engagement (but the released receptors are not detectable because of an
increased lability) was excluded by showing that receptor expression
measured by immunofluorescence flow cytometry remained unchanged over a
30-min period (data not shown). Consistent with these findings are the
results obtained with receptor-specific mAbs showing that mAb H398 (a
TNF-R55 specific and competing mAb) induces release of TNF-R75 (TNF-R55
could not be measured due to interference by the mAb with the ELISA)
while mAb utr-1 (a TNF-R75 specific and competing mAb) does not affect
release of TNF-R55 (TNF-R75 could not be measured due to interference
by the mAb with the ELISA). In additional experiments we observed that
Fab fragments prepared from the TNF-R55-specific and competing mAb
H398, which do not affect TNF receptors expression, completely
prevented TNF-induced down-modulation of both receptors (data not
shown).
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A panel of proteinase inhibitors was initially screened for their
effect on TNF-induced receptor solubilization. Table I shows that, among them, the
metalloproteinase inhibitors 1,10-phenanthroline and the
peptide-hydroxamates KB8301 and KB7785 strongly inhibited TNF-induced
release of both TNF receptors. The inhibitory effect of KB8301 and
KB7785 appears to be dependent on the hydroxamic acid-chelating moiety
since KB8845, which lacks this moiety, is ineffective. Shedding of
TNF-R55 was almost totally inhibited by KB8301 and 1,10-phenanthroline
and less markedly by KB7785. Shedding of TNF-R75 was less markedly
inhibited than shedding of TNF-R55 by all three compounds. The other
inhibitors tested were ineffective, with the possible exception of DFP,
which slightly but reproducibly inhibited TNF-R75 release only.
Hydroxamic acid-based compounds and 1,10-phenanthroline have been
extensively used to assess the involvement of metalloproteinases in
shedding of various receptors, including selectins, CD43, and TNF
receptors themselves (35, 36, 37) However, attention has not
been paid to the possible effects of these inhibitors on the
intracellular signaling pathways triggered by receptor engagement. We
therefore tested the effect of KB8301 and 1,10-phenantholine on
TNF-induced PMN activation as measured by the increase in
O2- production, adherence, and
expression of ß2 integrins. Table II shows that 1,10-phenanthroline
virtually abolishes TNF-induced cell activation, whereas KB8301 has no
effect. Similar results (i.e., strong inhibition with
1,10-phenanthroline or no effect with KB8301) were obtained after
stimulation with FMLP (data not shown). A strong inhibitory effect on
PMN activation was also observed using the nonchelating analogue
1,7-phenanthroline (data not shown). As these results point to a
nonspecific effect of 1,10-phenantroline, it was decided not to use
this inhibitor in subsequent studies.
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shows the dose-response curve of
the effect of KB8301, the most effective shedding inhibitor. From the
data of these experiments a ID50 values of 0.22
and 0.84 µM were calculated for TNF-R55 and TNF-R75, respectively.
The results of the figure also demonstrate that the less-pronounced
inhibition of TNF-R75 release by KB8301 cannot be overcome by
increasing its concentration. Fig. 5
shows that KB8301 also inhibits shedding of TNF-R55 and TNF-R75 induced
by FMLP, with a profile similar to that observed with TNF, i.e., almost
complete inhibition of TNF-R55 release, and partial inhibition of
TNF-R75 release.
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PMN contain a collagenase (MMP-8) and gelatinase B (MMP-9), two
zinc-dependent endopeptidases of the MMP family that are strongly
inhibited by peptide hydroxamates (38, 39, 40, 41). We used two
physiologic inhibitors of MMPs, i.e., TIMP-1 and TIMP-2
(42), to see if MMPs are involved in the shedding process.
Table III shows that neither inhibitor
affected TNF-induced receptor shedding. Since peptide hydroxamates are
known to inhibit zinc metalloproteinases (41), we tested
the effect of Zn2+ on inhibition of shedding by
KB8301. Using 0.1 mM excess ZnCl2 we were unable
to counteract the inhibitory activity of KB8301 (data not shown).
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shows that a substantial down-regulation of both TNF-R55 and TNF-R75
occurs in U937 cells after TNF treatment, indicating that a sheddase
similar to that functioning in PMN is operative in these cells, whereas
the expression of both receptors is not altered following incubation
with supernatants from either resting or FMLP-treated PMN. This
suggests that the proteinase is membrane bound, although we cannot
formally exclude that the inability to detect any shedding activity in
PMN supernatants might reflect the instability of the hypothetically
released sheddase, which might have completely lost its activity during
the period of time (about 5 min for cooling on ice and centrifugation)
needed to prepare the supernatants before assaying their activity,
which seems really very unlikely.
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The previous results indicate that a membrane-bound non-MMP is
involved in TNF-induced shedding of TNF receptors. Members of the ADAM
family of metalloproteinases, a class of membrane bound proteins that
contain both disintegrin and metalloproteinase domains, recently have
been shown to be involved in the shedding of the ectodomains of several
membrane proteins (43, 44). We have assessed the
expression in PMN of two of them known to possess proteinase
activity, i.e., ADAM-17 (TACE) and ADAM-12 (45). Fig. 6 shows that PMN express ADAM-17 to an
extent comparable to TNF receptors, whereas ADAM-12 was undetectable
using either mAbs, as shown in the figure, or polyclonal Abs (data not
shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). This finding differs from what previously
reported by Porteu and Hieblot (14) who showed that
TNF-induced down-modulation of its receptors from PMN is due to
shedding of TNF-R75 and internalization of TNF-R55. That
internalization of TNF-R55 takes place was, in fact, verified in our
laboratory too (Ref. 50 and data not shown), thus
indicating that both internalization and shedding are involved in the
down-modulation of this receptor. The failure by Porteu and Hieblot
(14) to detect shedding of TNF-R55 may well be explained
by the low sensitivity of their ELISA for the soluble receptor, which
may have led to underestimation of TNF-R55, the less abundant
(33) and less extensively shed (present paper) receptor.
Concerning the question regarding which of the two receptors mediates
shedding, the observations that TNF-R55-specific but not
TNF-R75-specific mAbs and mutants of TNF trigger release of both
receptors (Fig. 3) and that TNF-induced receptor release could be
inhibited by Fab fragments from a TNF-R55-specific and competing mAb,
categorically point to TNF-R55 as the receptor that transduces the
signal leading to shedding of both receptors. These results, combined
with the observations that all PMN responses to TNF so far studied are
mediated by TNF-R55, further emphasizes the notion that this receptor
is probably central to the modulation of all TNF-induced PMN
activities.
The nature of the proteinase involved in shedding is unknown. Data from
a study conducted with PMN militate against a role of serine or thiol
proteinases in this respect since inhibitors of these classes of
proteinases did not affect FMLP-induced shedding (11). In
cell systems different from PMN, hydroxamic acid-based compounds were
found to inhibit shedding of both TNF receptors (51, 52, 53),
suggesting that zinc-dependent metalloproteinases are involved in the
process, since hydroxamic acid-based compounds are potent inhibitors of
this class of proteinases (41). Our results (Table I) show
that TNF-induced shedding of its receptors is inhibited by the metal
chelators KB8301 and KB7785 but not by the metalloendopeptidase
inhibitors thiorphan and phosphoramidon or by EDTA. Insensitivity to
phosphoramidon and thiorphan indicates that membrane metalloproteases
of clan MA (54), which include the granulocyte Ags CD10
and CD13, have no role in the process. The lack of effect of EDTA,
which appears intriguing at first sight, will be discussed more in
detail below. KB8301 inhibited almost totally the release of TNF-R55
and, to a lesser degree, release of TNF-R75. In fact, 
40% of the
released TNF-R75 "evaded" inhibition at all inhibitor’s
concentrations (Fig. 4), suggesting that a proteinase insensitive to
this inhibitor contributes to shedding of this receptor. The
observation that among the other inhibitors used only DFP partially
inhibited TNF-R75 release suggests that this proteinase is a serine
proteinase. The identity of the proteinase, which accounts for the
DFP-inhibitable shedding upon TNF stimulation and which turns out to be
the responsible for the high levels of spontaneous (resting) release of
this receptor (Figs. 1 and 2) will be described elsewhere (C. Gasparini
and P. Dri, manuscript in preparation). Thus, a metalloproteinase is
involved in TNF-induced shedding of its receptors. The finding that
KB8301 caused an inhibition of FMLP-induced shedding similar to that
observed with TNF (Fig. 5) suggests that activation of a
metalloproteinase is also involved in receptor shedding induced by
ligands other than TNF.
Two sets of data help to better classify the enzyme responsible for the
stimulated release of TNF receptors in PMN. First, the results reported
in Table III which show that TIMP-1 and TIMP-2 do not affect
TNF-induced receptor shedding rule out the possibility that MMPs might
be involved in the process, and second, the failure to detect in
supernatants from FMLP-stimulated PMN any receptor releasing activity
(Table IV) suggests that the proteinase is membrane bound. Thus, the
involved sheddase is a non-matrix and membrane-anchored
metalloproteinase. These characteristics are distinctive for ADAMs, a
large group of metalloproteinases, included in family M12, subfamily B
(reprolysins) of clan MB, according to a recent classification
(55). A number of ADAMs have been identified and
molecularly cloned so far in mammals and many of them predicted to
encode potentially active metalloproteinases. However, few of them have
been shown to possess catalytic activity and to be expressed in
granulocytes. For example, ADAM-8 is expressed in granulocytes but has
no known catalytic activity (56). ADAM-9, which was shown
to be expressed in peripheral blood leukocytes by Northern blotting
(57), did not cleave a peptide spanning the cleavage site
of TNF-R55 and cleaved a TNF-R75 peptide at a site different from that
involved in receptor processing (58). ADAM-10, whose
presence in peripheral blood leukocytes has also been documented by
Northern blotting, was shown to possess pro-TNF- processing activity
and to cleave a peptide containing the proposed cleavage site of
pro-TNF-, but not peptides containing the proposed cleavage sites of
TNF-R55 or TNF-R75 (59). The recently described ADAM-12
(29) with functional proteolytic activity
(45) was also excluded as a possible candidate sheddase as
we could not find evidence of expression in PMN (Fig. 6). A remarkable
member of this family of metalloproteinases is ADAM-17. Initially
identified as the TACE (28, 60), it now appears to possess
a more relaxed specificity and is probably involved in the release of a
number of cytokines, growth factors and the corresponding receptors
initially synthesized as membrane-anchored proteins. For example, it
has been recently shown that cultured cells homozygous for a targeted
mutation in ADAM-17 were markedly deficient not only in the release of
TNF but also of TGF-, L-selectin, and TNF-R75 (44). The
presence of ADAM-17 in PMN has been demonstrated by Northern and
Western blotting (28) with an expression comparable to
that of TNF receptors, as shown in this paper (Fig. 6). Altogether,
these observations can be taken as a plausible circumstantial evidence
in favor of this metalloproteinase as the TNF receptor sheddase of
stimulated PMN. If this conclusion is correct, then the recently
published crystal structure of the catalytic domain of ADAM-17
cocrystallized with a hydroxamic acid inhibitor (61) may
provide useful hints to explain the two apparently incongruous results
obtained with the metalloproteinase inhibitors, i.e., the inability of
zinc to restore the activity inhibited by KB8301, and the lack of
effect of EDTA (Table I). Compound 3, the hydroxamic acid inhibitor
used in the above-mentioned study, has been shown to interact with the
catalytic site of ADAM-17 not only by chelation of
Zn2+ with the carbonyl and hydroxyl oxygens of
the hydroxamic acid moiety but also by forming hydrogen bonds and by
inserting an isobutyl residue into an hydrophobic pocket contained in
the active-site cleft. KB8301, the inhibitor used in the present study,
is very similar to compound 3 and is therefore expected to interact
with the catalytic site with high affinity, thus making it impossible
its displacement by Zn2+. This possibility is
substantiated by the observation that in the literature there are no
examples that we are aware, of a reversal by zinc of ADAMs activity
after inhibition by hydroxamic acid compounds.
The lack of effect of EDTA (Table I), which has a
pKa3 of 6.2 (62) and, therefore, is
expected to be highly polar at physiological pH, may be the consequence
of its inability to efficiently chelate the zinc contained in a
catalytic site with hydrophobic characteristics.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Pietro Dri, Department of Physiology and Pathology, University of Trieste, Via A. Fleming, 22, 34127 Trieste, Italy.
3 Abbreviations used in this paper: PMN, polymorphonuclear leukocytes; TNF-R55, 55-kDa TNF receptor; TNF-R75, 75-kDa TNF receptor; ADAM, a disintegrin and metalloproteinase; TACE, TNF- converting enzyme; MMP, matrix metalloproteinase; TIMP, tissue inhibitor of MMPs; DFP, diisopropylfluorophosphate; KB8301, [4-(N-hydroxyamino)-2R-isobutyl-3S-methylsuccinyl]-L-3-(5,6,7,8-tetrahydro-1-naphthyl)alanine-N-methylamide; KB8845, (4-hydroxy-2R-isobutyl-3S-methylsuccinyl)-L-3-(5,6,7,8-tetrahydro-1-naphthyl)alanine-N-methylamide; KB7785, [4-(N-hydroxyamino)-2R-isobutyl-3S-methylsuccinyl]-L-phenylglycine-N-methylamide; 125I-TNF, 125I-labeled TNF.
Received for publication December 15, 1999. Accepted for publication June 2, 2000.
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 E. C. Logue, S. Bakkour, M. M. Murphy, H. Nolla, and W. C. Sha ICOS-Induced B7h Shedding on B Cells Is Inhibited by TLR7/8 and TLR9 J. Immunol., August 15, 2006; 177(4): 2356 - 2364. [Abstract] [Full Text] [PDF]
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 J Plumb, S McQuaid, A K Cross, J Surr, G Haddock, R A. Bunning, and M N Woodroofe Upregulation of ADAM-17 expression in active lesions in multiple sclerosis Multiple Sclerosis, August 1, 2006; 12(4): 375 - 385. [Abstract] [PDF]
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 K. J. Garton, P. J. Gough, and E. W. Raines Emerging roles for ectodomain shedding in the regulation of inflammatory responses J. Leukoc. Biol., June 1, 2006; 79(6): 1105 - 1116. [Abstract] [Full Text] [PDF]
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A. S. Cowburn, A. Sobolewski, B. J. Reed, J. Deighton, J. Murray, K. A. Cadwallader, J. R. Bradley, and E. R. Chilvers Aminopeptidase N (CD13) Regulates Tumor Necrosis Factor-{alpha}-induced Apoptosis in Human Neutrophils J. Biol. Chem., May 5, 2006; 281(18): 12458 - 12467. [Abstract] [Full Text] [PDF]
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 V. Budagian, E. Bulanova, Z. Orinska, E. Duitman, K. Brandt, A. Ludwig, D. Hartmann, G. Lemke, P. Saftig, and S. Bulfone-Paus Soluble Axl Is Generated by ADAM10-Dependent Cleavage and Associates with Gas6 in Mouse Serum Mol. Cell. Biol., November 1, 2005; 25(21): 9324 - 9339. [Abstract] [Full Text] [PDF]
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V. Budagian, E. Bulanova, Z. Orinska, A. Ludwig, S. Rose-John, P. Saftig, E. C. Borden, and S. Bulfone-Paus Natural Soluble Interleukin-15R{alpha} Is Generated by Cleavage That Involves the Tumor Necrosis Factor-{alpha}-converting Enzyme (TACE/ADAM17) J. Biol. Chem., September 24, 2004; 279(39): 40368 - 40375. [Abstract] [Full Text] [PDF]
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W.-N. U. Chen, R. L. Woodbury, L. E. Kathmann, L. K. Opresko, R. C. Zangar, H. S. Wiley, and B. D. Thrall Induced Autocrine Signaling through the Epidermal Growth Factor Receptor Contributes to the Response of Mammary Epithelial Cells to Tumor Necrosis Factor {alpha} J. Biol. Chem., April 30, 2004; 279(18): 18488 - 18496. [Abstract] [Full Text] [PDF]
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N. Moghaddami, M. Costabile, P. K. Grover, H. P. A. Jersmann, Z. H. Huang, C. S. T. Hii, and A. Ferrante Unique Effect of Arachidonic Acid on Human Neutrophil TNF Receptor Expression: Up-Regulation Involving Protein Kinase C, Extracellular Signal-Regulated Kinase, and Phospholipase A2 J. Immunol., September 1, 2003; 171(5): 2616 - 2624. [Abstract] [Full Text] [PDF]
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J. Wang, R. S. Al-Lamki, H. Zhang, N. Kirkiles-Smith, M. L. Gaeta, S. Thiru, J. S. Pober, and J. R. Bradley Histamine Antagonizes Tumor Necrosis Factor (TNF) Signaling by Stimulating TNF Receptor Shedding from the Cell Surface and Golgi Storage Pool J. Biol. Chem., June 6, 2003; 278(24): 21751 - 21760. [Abstract] [Full Text] [PDF]
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 Y. Hirako, K. Yoshino, D. Zillikens, and K. Owaribe Extracellular Cleavage of Bullous Pemphigoid Antigen 180/Type XVII Collagen and Its Involvement in Hemidesmosomal Disassembly J. Biochem., February 1, 2003; 133(2): 197 - 206. [Abstract] [Full Text] [PDF]
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L. Y. Liu, J. B. Sedgwick, M. E. Bates, R. F. Vrtis, J. E. Gern, H. Kita, N. N. Jarjour, W. W. Busse, and E. A. B. Kelly Decreased Expression of Membrane IL-5 Receptor {alpha} on Human Eosinophils: II. IL-5 Down-Modulates Its Receptor Via a Proteinase-Mediated Process J. Immunol., December 1, 2002; 169(11): 6459 - 6466. [Abstract] [Full Text] [PDF]
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A. Krippner-Heidenreich, F. Tubing, S. Bryde, S. Willi, G. Zimmermann, and P. Scheurich Control of Receptor-induced Signaling Complex Formation by the Kinetics of Ligand/Receptor Interaction J. Biol. Chem., November 8, 2002; 277(46): 44155 - 44163. [Abstract] [Full Text] [PDF]
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N.-J. Chen, M.-W. Huang, and S.-L. Hsieh Enhanced Secretion of IFN-{{gamma}} by Activated Th1 Cells Occurs Via Reverse Signaling Through TNF-Related Activation-Induced Cytokine J. Immunol., January 1, 2001; 166(1): 270 - 276. [Abstract] [Full Text] [PDF]
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Y. Zhang, R. Guan, J. Jiang, J. J. Kopchick, R. A. Black, G. Baumann, and S. J. Frank Growth Hormone (GH)-induced Dimerization Inhibits Phorbol Ester-stimulated GH Receptor Proteolysis J. Biol. Chem., June 29, 2001; 276(27): 24565 - 24573. [Abstract] [Full Text] [PDF]
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C.-L. Tsou, C. A. Haskell, and I. F. Charo Tumor Necrosis Factor-alpha -converting Enzyme Mediates the Inducible Cleavage of Fractalkine J. Biol. Chem., November 21, 2001; 276(48): 44622 - 44626. [Abstract] [Full Text] [PDF]
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 J.-M. Fernandez-Real, B. Lainez, J. Vendrell, M. Rigla, A. Castro, G. Penarroja, M. Broch, A. Perez, C. Richart, P. Engel, et al. Shedding of TNF-alpha receptors, blood pressure, and insulin sensitivity in type 2 diabetes mellitus Am J Physiol Endocrinol Metab, April 1, 2002; 282(4): E952 - E959. [Abstract] [Full Text] [PDF]
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