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*
Department of Physiology and Pathology, University of Trieste, Trieste, Italy, and
Institute of Cell Biology and Immunology, University of Stuttgart, Stuttgart, Germany
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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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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Cytochrome c, type VI, from horse heart; elastase,
type VI, from porcine pancreas; BSA Fraction V, endotoxin tested and
cell culture tested; and FMLP were purchased from Sigma (St. Louis,
MO). Iodo-Gen (1,3,4,6-tetrachloro-3
,6-diphenylglycouril) was
from Pierce Chemical (Rockford, IL). Na125I was from
Amersham International (Little Chalfont, U.K.). Percoll was from
Pharmacia AB (Uppsala, Sweden). All solutions were made in
endotoxin-free water for clinical use.
Abs and cytokines
Purified mouse mAbs specific for TNF-R55 (mAb htr-9, an IgG1,
and mAb H398, an IgG2a) or TNF-R75 (mAb utr-1, an IgG1, and mAb 80 M2,
an IgG1) were either a generous gift of Dr. M. Brockhaus, Hoffmann La
Roche, Basel, Switzerland (mAbs htr-9 and utr-1) (3), or prepared as
described elsewhere (mAbs H398 and 80 M2) (14, 24). M80, a purified
rabbit polyclonal Ab specific for TNF-R75 was prepared as described
(24). Fab fragments derived from the polyclonal M80 were
prepared using the Immunopure Fab preparation kit (Pierce). Pure
recombinant human TNF, expressed in yeast, was obtained from Bissendorf
(Hannover, Germany). p55TNF and p75TNF, human TNF mutants with
exclusive specificity for TNF-R55 and TNF-R75, respectively (25), were
generously donated by Dr. H. Loetscher (Hoffmann La Roche). p55TNF
carries an Arg
Trp substitution at position 32 and a SerThr
substitution at position 86. p75TNF carries an AsnAsp substitution
at position 143 and an AlaArg substitution at position 145.
Neutrophil isolation
Blood was drawn from healthy volunteers and anticoagulated with 4 mM EDTA. Neutrophils were isolated according to a single-step separation procedure (26) with slight modifications. Blood (4 ml) was layered over a two-step Percoll gradient formed by 4 ml of 75% isotonic Percoll (75% Percoll, 10% PBS 10x, 15 ml of H2O, density (d) 1.103 g/ml, 300–310 mOsM) and 4 ml of 62% isotonic Percoll (62% Percoll, 10% PBS 10x, 28% H2O, d 1.078, 300–310 mOsM) in 15-ml conical test tubes. After centrifugation for 25 min (10 min at 200 x g and 15 min at 400 x g) at 20°C, the neutrophils, located at the interface between the two Percoll solutions, were collected, diluted in Ca2+- and Mg2+-free HEPES-buffered saline solution containing BSA (HBS--BSA, 145 mM NaCl, 5 mM KCl, 5 mM HEPES, 5 mM glucose, 0.2% BSA, pH 7.4), and centrifuged for 7 min at 200 x g. After elimination of contaminating erythrocytes by a brief hypotonic treatment (10-s incubation with 3 parts of a 1 mM sodium phosphate buffer solution, pH 7.4, followed by addition of 7 parts of 1.3% NaCl solution in 1 mM sodium phosphate buffer, pH 7.4, to restore isotonicity) and centrifugation, the cells were suspended in HBS--BSA. The resulting cell population contained 94–98% neutrophils, 2–5% eosinophils, and 1–2% mononuclear cells. The procedure was conducted at room temperature and in the absence of divalent cations to prevent neutrophil aggregation and activation. All subsequent experiments were conducted in HBS--BSA supplemented with 1 mM CaCl2 and 1 mM MgCl2 (HBS-BSA).
Assay of O2- production
Production of O2- by neutrophils residing on fibronectin-coated microtiter wells was measured by the superoxide dismutase-inhibitable cytochrome c reduction method, as described (18, 27).
Radioiodination of TNF
The Iodo-Gen method was used. Iodo-Gen, the oxidizing reagent, was dissolved in chloroform at a concentration of 50 µg/ml and 50 µl were added to a glass test tube. Just before the iodination was started, after the solvent was evaporated by a gentle flow of nitrogen, the tube was washed twice with PBS, and 2.5 µg of TNF in 25 µl of Ca2+- and Mg2+-free PBS were deposited at the bottom of the tube together with 2 µl of Na125I (0.2 mCi). The reaction was left to proceed for 8 min under gentle shaking and was stopped by the addition of 100 µl of a solution containing 100 µg/ml tyrosine in 1 mM NaI. After the addition of 200 µl of PBS containing 0.3% BSA, iodinated TNF was separated from free Na125I by gel filtration over a 5-ml column filled with Bio-Gel P-6 desalting gel (Bio-Rad, Richmond, CA). The two iodinations performed gave a 125I-TNF with estimated specific activities of 33 and 45 µCi/µg, respectively.
Receptor binding studies
Saturation binding experiments were conducted at 0–4°C to block release (shedding) or internalization of the receptors (28, 29, 30). PMN (5–10 x 106 cells/ml) in HBS-BSA were exposed to varying 125I-TNF concentrations (10–800 pM) in the absence and in the presence of an excess of cold TNF (200 times), to determine the nonspecific binding. To measure TNF-R55, mAb utr-1 at a concentration of 10 µg/ml was included in the incubation mixture to block binding of TNF to TNF-R75 (3, 31). To measure TNF-R75, mAb H398 was included in the reaction mixture to block binding of TNF to TNF-R55 (14). After 2.5 h of incubation on a rocking platform, unbound TNF was removed by centrifugation of the cell suspension for 30 s at 12,000 x g in a microfuge, followed by two washings with 0.75 ml of ice-cold HBS-BSA. The bottom parts of the conical microfuge tubes were cut off and counted in a gamma counter. A nonlinear regression analysis of the results was performed as described (32) by fitting the data to the equation of a binding isotherm (Langmuir isotherm) using the computer program Graphpad PRISM, version 2.0.
Determination of soluble TNF-R55 and soluble TNF-R75
A commercially available kit was used for these determinations (Amersham). The kit is based on an ELISA sandwich assay in which mAbs are used as capturing reagents and peroxidase-conjugated polyclonal Abs to detect binding to the capturing monoclonals.
Statistical analysis
Data were analyzed according to Student’s t test for paired data.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The expression on PMN of both types of TNF receptors, with TNF-R75
outnumbering TNF-R55, has been reported in most published studies (3, 5, 18, 29), but their binding characteristics in these cells are not
known. Table I
shows that PMN express
more than twice as much TNF-R75 as TNF-R55, thus confirming the data of
the majority of previous studies, and that the affinity of TNF-R75 for
TNF is higher than that of TNF-R55, in our experimental conditions, in
agreement with what has been reported in other cell systems (4, 6, 33, 34). In spite of its lower degree of expression, TNF-R55 is considered
the exclusive signaling receptor in the TNF-induced activation of
respiratory burst and other PMN responses. This notion derives from the
results of studies with receptor-specific mAbs showing that
anti-TNF-R55 mAbs are on their own agonistic and that their Fabs
inhibit the responses to the cytokine, while anti TNF-R75 mAbs do not
exert any effects (18, 19, 20). To verify this conclusion more directly, we
studied the effects that recombinant mutants of TNF, which specifically
recognize TNF-R55 (p55TNF) or TNF-R75 (p75TNF), exert on the
respiratory burst of PMN adherent to fibronectin-coated surfaces,
assayed as O2- production. Fig. 1 shows that: 1) p55TNF, but not p75TNF,
stimulates O2- production by the adherent PMN;
2) the response to p55TNF is lower than that to TNF; and 3) p55TNF and
p75TNF given in combination do not induce a response greater than that
induced by p55TNF alone. The response to p55TNF is dose dependent and
plateaus at concentrations above 15 ng/ml. The lower response to p55TNF
with respect to TNF cannot be attributed to differences in affinity for
the receptor since TNF and p55TNF have a similar
KD for TNF-R55 (25). Concentrations of p75TNF 3
times as high as those used in Fig. 1 were also ineffective (data not
shown), making it unlikely that the lack of response to p75TNF is due
to an affinity for the receptor lower than that of TNF (25). Table II shows that both a monoclonal (utr-1)
and a polyclonal (M80) TNF-R75-specific and ligand-competing Ab inhibit
the production of O2- induced by TNF
suggesting that TNF-R75 is required for maximal response to TNF. Table II also shows that a similar inhibition is observed using the Fab
fragments of M80, indicating that the inhibitory effect of the Abs is
not due to receptor cross-linking. Table III shows that neither mAb utr-1 nor the
polyclonal Ab M80 and its corresponding Fab fragments influence the
response induced by TNF-R55-specific agonists, i.e., p55TNF and mAbs
htr-9 and H398, indicating that their inhibitory effect is not due to
interference with TNF-R55 signaling, and hindrance of TNF binding
remains the more likely possibility. Another TNF-R75 specific mAb (mAb
80M2), which at variance with the anti-TNF-R75 Abs utr-1 and M80
does not compete for TNF binding also inhibits the response to TNF but
not the response to the TNF-R55-specific agonists p55TNF and mAb H398
(Table IV). This mAb increases the
affinity of TNF-R75 for TNF by more than one order of magnitude by
strongly decreasing the dissociation rate (Koff)
of TNF (35). These results suggest that cooperation by TNF-R75 requires
not only TNF binding to the receptor but also an appropriate
dissociability from it.
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The results of the previous section show that TNF-R75 cooperates
with TNF-R55, the signaling receptor, for maximal response to TNF. Fig. 2 shows that inhibition of the
O2- production by PMN exposed to varying TNF
doses by the TNF-R75-specific and ligand-competing mAb utr-1 becomes
more prominent at decreasing TNF concentrations, indicating that the
contribution of TNF-R75 might be particularly important for the
response to physiologic TNF concentrations. It might be argued that the
lower inhibition observed at increasing TNF doses is due to an
ineffective competition of mAb utr-1 with TNF for binding to TNF-R75.
Two sets of data speak against this possibility: 1) the strength of
binding of utr-1 for TNF-R75 appears much greater than that of TNF as
indicated by experiments showing that TNF bound to the receptor is
easily removed by a brief acid wash at pH 3, while mAb utr-1 is not
(data not shown); and 2) utr-1 concentrations 2 or 3 times higher than
those used in the experiments of Fig. 2 gave an inhibition of the
TNF-induced respiratory burst comparable with that observed at lower
concentrations (data not shown). The data obtained with mAb utr-1 are
further supported by the results of experiments with mAb 80 M2 which,
as already noted before, does not compete for TNF binding to TNF-R75
but decreases its dissociation from the receptor (Fig. 3). Fig. 3 in fact shows that, even with
this mAb, inhibition of the respiratory burst increases by decreasing
TNF concentrations.
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shows that the response to TNF,
when its binding to TNF-R75 is prevented by mAb utr-1, becomes
indistinguishable from the response to p55TNF, further emphasizing the
concept that signaling via TNF-R55 is maximal when both TNF receptors
are simultaneously engaged by the cytokine.
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Given the results of the previous sections, it is reasonable to
predict that cells with reduced expression of TNF-R75 would mount a
lower response to the cytokine. To this end we used elastase, an enzyme
known to specifically cleave TNF-R75 from neutrophils (5). Fig. 5 shows that, indeed, elastase
down-modulates the expression of TNF-R75 (Fig. 5A), that
this is accompanied by release of the soluble form of the receptor
(Fig. 5B), and that this effect is specific for TNF-R75
since expression of TNF-R55 as well as release of its soluble form
remain unaffected. Fig. 6 shows that
elastase-treated PMN, on exposure to TNF, produce less
O2- than control PMN. The difference in
response between elastase-treated cells and control cells is observed
at all TNF concentrations, although statistical significance could be
demonstrated only at 0.5 and 5 ng/ml TNF. The lack of effect of the
elastase treatment on the response to 20 and 40 ng/ml TNF is not
surprising if one considers that the cooperation of TNF-R75 to TNF-R55
signaling is more pronounced at low cytokine doses (see previous
paragraph) and that elastase-treated cells have lost only part of
TNF-R75 (Fig. 5), which may not be sufficient to alter the response to
high TNF doses.
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shows that elastase treatment does not
affect the O2- generation induced by
TNF-R55-specific agonists, i.e., p55TNF and mAb H398, or
FMLP, indicating that the enzyme did not harm the TNF-R55 and the FMLP
receptor or the signaling cascade downstream of the two receptors or
the O2--generating enzyme complex.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Evidence thus far available in favor of TNF-R55 as the signaling receptor for PMN respiratory burst derives from experiments using agonistic and antagonistic receptor-specific monoclonal and polyclonal Abs (18, 19, 20). This seems conclusively confirmed by the results reported in the present paper obtained by a more direct approach that uses recently developed reagents, i.e., TNF mutants specific for TNF-R55 or TNF-R75. This conclusion is consistent with the results reported by others showing that TNF-R55-specific TNF mutants mediate several neutrophil responses including Ab-dependent cell cytotoxicity, priming for FMLP-induced O2- production and cytotoxicity and, in addition, induce inflammation and neutrophil infiltration in baboons, whereas TNF-R75-specific TNF mutants are devoid of any of such activities (22, 43).
Our results clearly show that TNF-R75 cooperation is required for an
optimal response to TNF to take place. In fact, 1) none of the
TNF-R55-specific agonists (p55TNF and mAbs) were able to induce a
respiratory burst comparable with that induced by TNF, 2) the response
to TNF was decreased either by blocking its binding to TNF-R75 (Table II
) or by increasing its strength of association with the receptor
(Table IV) using monoclonal or polyclonal Abs, and 3) PMN with a
decreased expression of TNF-R75 showed a diminished response to the
cytokine (
Figs. 5–7).
The cooperative role of TNF-R75 is more remarkable at low TNF doses, as
indicated by the observations that inhibition by TNF-R75-specific mAbs
increases by decreasing the concentrations of the cytokine (Figs. 2 and 3) and that the diminished response of elastase-treated PMN is
particularly evident at low TNF concentrations (Fig. 6). This suggests
that TNF-R75 might play an important modulatory role in TNF-induced
respiratory burst at physiologically relevant concentrations of the
cytokine.
Our results strongly argue against the possibility that the cooperation
between the two receptors might take place at the signaling level,
i.e., that occupancy of the TNF-R75 might activate a signaling pathway
independently of TNF-R55. In fact, the combination of p55TNF and p75TNF
does not result in a response greater than that induced by p55TNF alone
(Fig. 1); neither was the response to p55TNF increased by p75TNF in the
presence of mAb 80 M2, which strengthens the interaction of p75 TNF
with TNF-R75 (data not shown).
Two explanations have been offered thus far in the literature for the cooperation between the two TNF receptors observed in other experimental sets: 1) formation of TNF-R55/TNF-R75 heterocomplexes; and 2) the ligand passing model.
TNF-induced formation of heterocomplexes consisting of both TNF-R55 and TNF-R75 has been shown to occur in vitro in cultured and primary murine cells (44). It has also been demonstrated that one of the signal-transducing proteins that interact with TNF-R75, i.e., TNF-R75-associated factor 2 (TRAF2) can also interact with TNF-R55-associated death domain protein (TRADD) bound to the intracellular moiety of TNF-R55 (45). Therefore, it is conceivable that the TNF-R55/TNF-R75 complexes potentiate signaling via TNF-R55 by facilitating the association of TNF-R75 and TNF-R55 signal transducers, through juxtaposition of intracellular tails of the receptors. We tried to verify the heterocomplex hypothesis by comparing the respiratory burst of PMN in which the two TNF receptors were cross-linked separately, or jointly. As expected, addition of a second anti-mouse polyclonal Ab to PMN preincubated with the Fab fragments from an anti-TNF-R55 mAb (H398), but not to PMN preincubated with an anti-TNF-R75 mAb (utr-1) stimulated O2- production. When the second Ab was added to PMN preincubated with both the anti-TNF-R55 Fabs and the anti-TNF-R75 mAb, the response was lower than that induced by cross-linking the TNF-R55 alone (data not shown). These findings seem to speak against a cooperation between the two TNF receptors based on heterocomplex formation, although they cannot be taken as a conclusive evidence. In fact, the heterocomplexes formed after Ab cross-linking may contain the two TNF receptors in proportions different from those found in the presence of TNF, which would preclude any direct comparison between the two conditions.
According to the ligand passing model which has been proposed on the
basis of binding data obtained at 0–4°C (46), TNF-R75, the higher
affinity receptor (4), would catch TNF and deliver it to TNF-R55 and in
this way enhance binding to and signaling through TNF-R55. This model
encounters some difficulties with the thermodynamics. How can a ligand
move from a high affinity to a low affinity receptor? Why should TNF
pass to the low affinity receptor rather than reassociate with the same
receptor? Where does the energy required come from? Recent published
data showing that at 37°C, at variance with what observed at
0–4°C, TNF-R55 is the higher affinity receptor (47) appear to
overcome some of the above difficulties. However, even in this case, a
question remains open and that is whether TNF-R-75, the low affinity
receptor, may bind enough TNF to explain its cooperation with TNF-R55,
according to the passing model. Reasoning only in terms of affinity is
not helpful in this respect since the concept of affinity refers to
equilibrium conditions and does not take into account the dynamics of
the interaction, which may be critical for subsequent events to occur.
Thus, on the basis of the data obtained at 37°C (47) and of the
equations reported in Ref. 32, we have determined some kinetic
parameters i.e., Kobs,
t1/2(ass), t1/2(diss) and
receptor saturation (RLeq) for a TNF
concentration of 0.5 ng/ml (10-11 M). This concentration
was chosen because cooperation by TNF-R75 is more prominent at low TNF
doses. What the calculated effective association rate constants
(Kobs) tell us is that TNF associates 20 times
more rapidly with TNF-R75 (Kobs = 0.645
min-1) than with TNF-R55 (Kobs =
0.032 min-1). Association to TNF-R55 is rather slow
(t1/2(ass) = 21.6 min) and reaches 34%
saturation at equilibrium (RLeq = 0.34). For
TNF-R75, equilibration is more rapid
(t1/2(ass) = 1.07 min) reaching, however,
only 2.3% saturation (RLeq = 0.023).
Accordingly, these few TNF-R75 complexes should dissociate quite
rapidly to feed significantly the TNF-R55 pool. Given that
t1/2 for dissociation of TNF-R75
complexes is 1.1 min, it turns out that in 
10 min 184 TNF molecules
should exchange (802 TNF-R75/cell, 2.3% saturation, 10 times an
exchange). Since this figure accounts for >50% of the available
TNF-R55 (see Table I), it is reasonable to believe that ligand passing
may significantly contribute to the response. Once formed, TNF/TNF-R55
complexes appear to have an extraordinary stability
(t1/2(diss) = 33.2 min) which
would favor the association with the cytoplasmic tail of the
oligomerized receptor of transducing molecules known to be involved in
signaling via this receptor (48).
The ligand passing model does not exclude that receptor heterocomplexes may form, which could represent a potential transient intermediate in the passing process.
In conclusion, we have shown that the participation of TNF-R75 is necessary for an optimal TNF-induced activation of the respiratory burst and discussed the possible mechanisms of action. Whether TNF-R75 only serves as a modulator of the respiratory burst or mediates other PMN responses to TNF is not known.
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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. E-mail address:
3 Abbreviations used in this paper: PMN, polymorphonuclear leukocytes; TNF-R55, 55-kDa TNF receptor; TNF-R75, 75-kDa TNF receptor; p55TNF, TNF mutant specific for TNF-R55; p75TNF, TNF mutant specific for TNF-R75; O2-, superoxide anion; HBS-, HEPES-buffered saline solution containing BSA; TRADD, TNF-R55-associated death domain protein.
Received for publication February 25, 1998. Accepted for publication September 15, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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(TNF-)-induced cell adhesion to human endothelial cells is under dominant control of one TNF receptor type, TNF-R55. J. Exp. Med. 177:1277. (TNF)-induced activation of neutrophil respiratory burst on biologic surfaces is mediated by the p55 TNF receptor. Blood 84:287. on superoxide generation in human neutrophils. Biochem. J. 298:733.
cytotoxic and proinflammatory activities by p55 receptor- and p75 receptor-selective TNF- mutants. EMBO J. 13:843.[Medline]
(TNF) on biologic surfaces. J. Cell Biol. 135:511. (TNF) mutants with exclusive specificity for the 55-kDa or 75-kDa TNF receptors. J. Biol. Chem. 268:26350. is not needed for induction of a biological effect via TNF receptors. J. Biol. Chem. 265:22409.
B activation prevents cell death. Cell 87:565.[Medline]
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) and of receptor-specific TNF mutants p55TNF
(
), p75TNF (
), and p55TNF + p75TNF (
). PMN suspensions
(1.5 x 106/ml in HBS), after temperature
equilibration at 37°C for 10 min were added in 50-µl aliquots to
the wells for the assay of O2- production. A
representative experiment of four similar ones, each conducted in
duplicate, is reported.
