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Endotoxin-Stimulated Monocytes Release
Multiple Forms of IL-1 β, Including a ProIL-1
β Form Whose Detection Is Affected by
Export
Mark D. Wewers2, Alissa V. Winnard and Heidi A. Dare
J Immunol 1999; 162:4858-4863; ;
http://www.jimmunol.org/content/162/8/4858
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Copyright © 1999 by The American Association of
Immunologists All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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References
Endotoxin-Stimulated Monocytes Release Multiple Forms of
IL-1b, Including a ProIL-1b Form Whose Detection Is
Affected by Export1
Mark D. Wewers,2 Alissa V. Winnard, and Heidi A. Dare
nterleukin-1b is a potent proinflammatory cytokine regulated
at many sites in its induction, production, and function. Its
induction by endotoxin is modified by the concentration of
LPS-binding protein, soluble and surface CD14 expression, and
factors that control endotoxin tolerance (1–5). Once induced, its
gene expression is regulated by both transcriptional and translational controls (6 –10). Once released, mature IL-1b is antagonized
by IL-1ra and the soluble form of the type II IL-1R (11–15). However, IL-1b is most tightly regulated in processing and release (10,
16, 17). This aspect of regulation is possibly the least understood.
IL-1b is translated as a 31-kDa molecule that lacks a conventional leader sequence or membrane-spanning region (18). It is
synthesized in the cytosol on free polyribosomes (19), and its conversion to mature IL-1b requires processing by a cysteine protease
termed IL-1b-converting enzyme (ICE),3 or more recently caspase
1 (20 –22). Like proIL-1b, ICE is also found in the cytosol as an
inactive precursor protein (23). Two 45-kDa ICE precursor molecules must be processed at specific aspartic acid-X sites to form
homodimers that condense to form the functional tetramer (24,
25). While ICE is assumed to be the IL-1b convertase, active ICE
has yet to be convincingly identified in cells that process and release IL-1b (23, 26). Furthermore, the connection between
proIL-1b conversion to the mature 17-kDa IL-1b and its release
from the cytosol is uncertain.
I
It has been suggested that released proIL-1b may be subject to
processing (and hence activation) extracellularly (27). Furthermore, using a synthetic ICE inhibitor, it has been shown that
proIL-1b processing is not a prerequisite for IL-1b export from the
cell (20). In this context, components of IL-1b distinct from the
17-kDa mature form are released from intact mononuclear phagocytes (28). The present study demonstrates that IL-1b is released
in a number of different forms, one of which is proIL-1b, which
differs significantly from cytosolic proIL-1b in its ability to be
recognized by Abs and the type II IL-1R.
Materials and Methods
Purification of PBMC and cell culture conditions
Venous blood was obtained from healthy normal volunteers by venipuncture. The blood was anticoagulated with sodium heparin (Elkins-Shinn,
Cherry Hill, NJ) at 15 U/ml and kept at 4°C during processing. PBMC were
separated by polysucrose/sodium diatrizoate (Histopaque; Sigma Diagnostics, St. Louis, MO) density-gradient centrifugation, washed three times
with sterile saline, and then resuspended at 5 3 106 cells/ml in RPMI 1640
(BioWhittaker, Walkersville, MD) and supplemented with 5% FBS (HyClone, Logan, UT) and gentamicin (50 mg/ml), unless otherwise noted.
PBMC were cultured in either 6- or 24-well tissue culture plates (Becton
Dickinson, Lincoln Park, NJ) at 5 3 106 cells/ml and stimulated with LPS
at 1 mg/ml (LPS W, Escherichia coli 102F:B8; Difco, Detroit, MI) at 37°C,
5% CO2.
Preparation of [35S]methionine-labeled released and cytosolic
IL-1b
Department of Pulmonary and Critical Care Medicine, Ohio State University, Columbus, OH 43210
Received for publication February 11, 1998. Accepted for publication January
14, 1999.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
Supported by National Institutes of Health Grant HL40871 (to M.D.W.).
2
Address correspondence and reprint requests to Dr. Mark D. Wewers, N325 Means
Hall, 1654 Upham Drive, Columbus, OH 43210. E-mail address: [email protected]
3
Abbreviation used in this paper: ICE, IL-1b-converting enzyme.
Copyright © 1999 by The American Association of Immunologists
IL-1b preparations were metabolically labeled with [35S]methionine by
culture in methionine-free RPMI 1640 and the addition of 0.5 mCi/ml
[35S]methionine (Amersham, Arlington Heights, IL) with the endotoxin.
After 18 h, culture supernatants and cells were harvested and prepared for
immunoprecipitation. Cells were lysed in a buffer containing 10 mM Tris,
pH 7.4, 1% Nonidet P-40 (Sigma), 1 mg/ml leupeptin, 1 mg/ml pepstatin,
1 mM EDTA, and methoxysuccinyl-ala-ala-pro-val chloromethyl ketone.
Lysates were cleared of nuclei and cell particulate by centrifugation at
1000 3 g.
Supernatants and lysates were buffered to pH 8 with 1 M Tris and then
incubated with either carboxyl terminus-specific (Rc) or amino terminusspecific (Rn) rabbit antisera to human IL-1b at 1/100 dilution at 4°C for
0022-1767/99/$02.00
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The processing and release of 31-kDa proIL-1b to the mature 17-kDa form of IL-1b are still poorly understood. To help elucidate
the mechanisms involved in IL-1b processing and release, we measured IL-1b forms released from endotoxin-stimulated monocytes by immunoprecipitation of [35S]methionine-labeled protein, by Western blots, and by our recently developed ELISA specific
for proIL-1b. Our studies demonstrate that in addition to the 17-kDa mature IL-1b, IL-1b is also released as 31-, 28-, and 3-kDa
molecules. The 31-kDa-released form of proIL-1b represented 20 – 40% of the total released IL-1b, as measured by SDS-PAGE
with densitometry. This released proIL-1b was susceptible to ICE processing; however, this proIL-1b was not detectable by either
a mature or proIL-1b-specific ELISA, suggesting that release induces a conformational change. The ELISA inability to detect
proIL-1b was not due to inadequate sensitivity or subsequent degradation in the ELISA. Furthermore, while immunoaffinitypurified cytosolic proIL-1b could complex the type II IL-1R, released proIL-1b did not. Finally, the absence of a band shift in
nondenaturing gel electrophoresis excluded proIL-1b binding to another protein. These findings imply that IL-1b is exported from
monocytes as 3-, 17-, 28-, and 31-kDa forms and that the released 31-kDa form differs from cytosolic proIL-1b. The Journal of
Immunology, 1999, 162: 4858 – 4863.
The Journal of Immunology
1 h. Ig-complexed material was incubated with protein A-Sepharose (BioRad Laboratories, Hercules, CA) for 1 h, and unbound material was removed by washing three times with PBS. The beads were eluted with
100 mM glycine buffer, pH 3, and the eluate was buffered immediately to
pH 7 with 1 M Tris.
IL-1b enzyme-linked immunoassays
Western blot analysis
Immunoblots of cell supernatants (106 cells/ml) and lysates (107 cells/ml)
were used to assay mononuclear phagocytes for endogenous cytokine expression. Samples were boiled for 3 min in equal volume of Laemmli
sample buffer and electrophoresed into a 15% SDS-polyacrylamide gel,
transferred to Immobilon P membranes (Millipore, Bedford, MA) in Tris/
10% methanol/0.01% SDS buffer, air dried, and then blocked and assayed
by a lumiphos technique per manufacturer’s recommendation (Amersham).
ProIL-1b was detected using a 1/100 dilution of rabbit antiserum to
proIL-1b (Rn).
Bronchoalveolar lavage
Bronchoscopy with bronchoalveolar lavage was performed, as we have
previously described (10). Briefly, subjects underwent standard bronchoscopy. The bronchoalveolar lavage consisted of instilling sequentially aspirating sterile saline in five 20-ml aliquots into the right middle or lingular
bronchus from the wedged position. Recovered lavage fluid was passed
through one layer of sterile surgical gauze to remove mucus and particulate. Cells were counted by hemocytometer, pelleted, and resuspended in
RPMI 1640 for in vitro studies.
ICE processing of proIL-1b
The ability of proIL-1b to be processed by ICE was evaluated by incubating immunoaffinity-purified [35S]methionine-labeled proIL-1b with 5 U
functional rICE (gift of Nancy Thornberry and Doug Miller, Merck Research Laboratories, Rahway, NJ) in 25 ml vol of processing buffer (50 mM
HEPES, pH 7.5, 10% sucrose, 0.1% CHAPS, 2 mM DTT) for 2 h at 30°C.
Proteolysis was stopped by the addition of 1 mM iodoacetate. Processed
proIL-1b was evaluated by subsequent SDS-PAGE and autoradiography
for generation of the appropriately sized proIL-1b fragments.
Another approach to document proIL-1b processing was to quantify
samples by both the mature IL-1b ELISA (B1/Rc) and the proIL-1b
ELISA (Rn/G). ICE processing of proIL-1b was characterized by gain of
B1/Rc detection and loss of Rn/G detection.
for 1 h at 20°C, a nonblocking mAb to the IL-1R (M2) (gift of Michael
Widmer and Steven Dower) was then incubated at 5 mg/ml for 1 h at 4°C,
and then protein G-Sepharose beads were added for an additional hour.
After washing three times, the beads were boiled in Laemmli sample buffer
and subjected to 12% SDS-PAGE. Gels were fixed with Enhance (Amersham), and autoradiography was performed.
Type II IL-1R binding of released and cytosolic proIL-1b was also
studied using a modification of the capture format described by Slack et al.
(13). Briefly, Immunolon IV plates (Dynatech, McLean, VA) were coated
overnight with the nonneutralizing mAb to the type II IL-1R, M2 (1 mg/
well), washed, and then incubated with 10 ng/ml of recombinant type II
IL-1R. Immobilized type II receptor was then incubated 1 h with recombinant mature and proIL-1b standards, or with unlabeled, endotoxin-stimulated PBMC cytosol or supernatants (proIL-1b was documented by both
Rn/G ELISA and Western blotting with Rn antisera). After washing, bound
IL-1b and proIL-1b were detected by Rc and Rn antisera, respectively, and
peroxidase-conjugated goat anti-rabbit antisera and o-phenylenediamine,
as outlined for ELISA formats above.
Results
High m.w. protein coprecipitates with IL-1b in mononuclear
phagocyte supernatants
While characterizing the processing and release of mature IL-1b
by mononuclear phagocytes, we noted that endotoxin-stimulated
monocyte supernatants also contain a 31-kDa protein that coincides with the size of intracellular proIL-1b. Metabolic labeling
with [35S]methionine, followed by immunoprecipitation and SDSPAGE, revealed that both human blood monocytes and alveolar
macrophages released what appeared to represent 31-kDa IL-1b.
The kinetics of the high m.w. protein release paralleled the release
of 17-kDa mature IL-1b (10).
Demonstration that the high m.w. factor is proIL-1b
To determine whether the 31-kDa protein was proIL-1b, selective
immunoprecipitation experiments were performed to further characterize this protein. Fig. 1 compares the relative ability to immunoprecipitate this protein by a rabbit antiserum specific to the
amino terminus of proIL-1b (Rn) and a carboxyl terminus-specific
rabbit antiserum (Rc). Both the Rn and Rc antisera were able to
immunoprecipitate the 31-kDa molecule, as would be expected for
proIL-1b. Furthermore, the Rn antiserum also immunoprecipitated
a 3-kDa fragment of IL-1b (the expected product of cleavage at
Asp28-X) (30), while the Rc antisera immunoprecipitated the carboxyl-terminal fragment of this processing, 28-kDa IL-1b. Consistent with this observation, the Rn antisera did not detect the 28or 17-kDa fragments, and the Rc antisera did not detect the 3-kDa
fragment.
To confirm that the 31-kDa protein immunoprecipitated by Rn
was proIL-1b, the immunoprecipitation was repeated in the presence or absence of the peptide that had been used to generate the
Rn antisera. Fig. 2 documents the specificity of the 31-kDa protein
as IL-1b by demonstrating that the immunizing peptide blocked
the Rn antisera detection of the 31-kDa protein, but not the Rc
antisera detection of either the 31- or 17-kDa proteins. The immunizing peptide also blocked the detection of the 3-kDa fragment
(data not shown), confirming its identity as a precursor fragment.
Binding of proIL-1b to type II IL-1R
Ability of IL-1-converting enzyme to process released proIL-1b
Binding of proIL-1b to the type II IL-1R was evaluated by two techniques.
The first tested the ability of the soluble recombinant type II IL-1R to
immunoprecipitate proIL-1b, and the second utilized the type II IL-1R in
a capture ELISA format.
Recombinant soluble type II receptor (gift of Michael Widmer and
Steven Dower, Immunex, Seattle, WA) at 10 ng/ml was incubated with the
cytosol or supernatant samples of endotoxin-stimulated PBMC, which had
been generated with [35S]methionine, as outlined above. After incubation
One of the characteristics of proIL-1b is its ability to be processed
by ICE to functional 17-kDa IL-1b. The ability of the released
proIL-1b to undergo activation by ICE is particularly relevant to
understanding the significance of the released proIL-1b. To test
this question, released and cytosolic proIL-1b were labeled metabolically with [35S]methionine in the same individuals and purified by affinity chromatography. The released and cytosolic
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Three distinct IL-1b ELISA formats were utilized. Mature IL-1b was detected using a sandwich ELISA format that we have previously described
(10). This ELISA, termed B1/Rc, utilizes a mouse mAb to mature human
IL-1b, B1 (gift of Ann Berger, Upjohn, Kalamazoo, MI), as the capture, a
rabbit polyclonal antisera to mature human IL-1b (Rc) (generated in our
laboratory) to complete the sandwich, and a peroxidase-conjugated goat
anti-rabbit antiserum for detection by the substrate o-phenylenediamine
(Sigma). Since the B1 capture Ab blocks IL-1b function, this ELISA format does not recognize IL-1b if the functional site is unavailable.
ProIL-1b was detected by two different proIL-1b-specific ELISAs. The
first was used as we previously described (29). This ELISA is identical to
the mature ELISA with the exception that Rc, the carboxyl terminus-specific antiserum, is replaced with Rn, a proIL-1b-specific rabbit antiserum
generated against a synthetic peptide corresponding to amino acids 3–21 of
human proIL-1b. This ELISA, termed B1/Rn, has documented specificity
for proIL-1b and a sensitivity to 100 pg/ml (29).
A second proIL-1b-specific ELISA was developed that did not require
the availability of the functional B1 epitope. This ELISA utilizes the amino
terminus-specific antiserum, Rn, as the capture Ab, and a goat anti-human
IL-1b Ab (R&D Systems, Minneapolis, MN) to complete the sandwich. A
horseradish peroxidase-conjugated rabbit anti-goat Ab, followed by
o-phenylenediamine, was used to detect the bound goat Ab. This ELISA,
termed Rn/G, was confirmed to be specific for proIL-1b, as it did not detect
mature IL-1b and was sensitive to 100 pg/ml.
4859
4860
RELEASED FORMS OF IL-1b
proIL-1b were then tested for their ability to be processed by functional ICE. Fig. 3 shows a representative experiment. Both released proIL-1b and cytosolic proIL-1b are processed to the 28and 17-kDa forms. This characteristic cleavage pattern further documents the identity of the proIL-1b and suggests the possibility
that the release form of proIL-1b may be subsequently processed
extracellularly.
Released proIL-1b differs from cytosolic proIL-1b
Detection by proIL-1b ELISA and immunoblotting. Using the
proIL-1b-specific ELISA (B1/Rn) (29), we attempted to characterize the kinetics of the release of proIL-1b by stimulated blood
monocytes. Unexpectedly, although the proIL-1b ELISA can detect both recombinant and cytosolic forms of proIL-1b with a sensitivity of at least 100 pg/ml, little to no released proIL-1b could
be detected using this ELISA (data not shown). In contrast, the
released form of proIL-1b was readily detectable by Western blotting (Fig. 4). Semiquantitative analysis of the immunoblots by densitometry using a recombinant proIL-1b standard showed released
FIGURE 2. Ability of immunizing peptide 3–21 to block the immunoprecipitation of the release 31-kDa form. Supernatant from [35S]methionine-labeled mononuclear cells after overnight culture with 1 mg/ml LPS
was incubated with (1) or without (2) peptide 3–21 (1 mg/ml) just before
the immunoprecipitation with either Rn or Rc antisera, as outlined in Fig.
1. Shown is the autoradiogram from the 12% SDS-PAGE of the immunoprecipitates. It should be noted that the 3-kDa IL-1b fragment was visible
on other repeat experiments with Rn and its immunoprecipitation was also
blocked by peptide 3–21.
proIL-1b present at about 25% of the amount in lysates (i.e., 1
ng/ml range).
Since it was conceivable that proIL-1b might undergo partial
processing during the ELISA assay (and therefore prevent detection by loss of the Rn epitope), purified [35S]methionine-labeled
cytosolic proIL-1b was tested for its stability in the ELISA format.
The ELISA-immobilized proIL-1b was not cleaved, as documented by SDS-PAGE and autoradiography (data not shown).
Finally, since the B1/Rn ELISA format utilizes mAb B1 as the
capture Ab, it was conceivable that the B1 epitope is unavailable
in the released form of proIL-1b, but available in the recombinant
proIL-1b standard and cytosolic proIL-1b. Consistent with this
hypothesis, a unique proIL-1b ELISA format (Rn/G), which utilizes the rabbit proIL-1b-specific antisera (Rn) as the capture Ab
and a polyclonal goat Ab (G) to mature IL-1b to complete the
sandwich, was able to detect released proIL-1b. Table I compares
the detection of released proIL-1b by the two ELISA formats,
FIGURE 3. Ability of released proIL-1b to be processed by IL-1b-converting enzyme. [35S]Methionine-labeled proIL-1b was obtained from the
cytosol (lys.) or supernatant (sup.) of endotoxin-treated PBMC by immunoprecipitation with Rn antisera, as previously described. Eluted proIL-1b
was subjected to 5 U active rICE in 25 ml ICE buffer for 2 h at 30°C.
Proteolysis was stopped by 1 mM iodoacetate, and resulting products were
analyzed by 12% SDS-PAGE and autoradiography. This is representative
of three experiments. The nonspecific high m.w. forms in the supernatant
immunoprecipitations were also seen with preimmune antisera (see Fig. 1).
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FIGURE 1. Immunoprecipitation of released forms of IL-1b. The left side outlines the m.w. forms of IL-1b expected from the processing of proIL-1b
at site 1 (Asp28) and site 2 (Asp116). The ability of either the Rc (carboxyl terminus-specific rabbit polyclonal Ab to mature IL-1b) or the Rn (amino
terminus-specific polyclonal antiserum to amino acids 3–21 of proIL-1b) to recognize the various forms is denoted by a (1). The right side of the graphic
shows a representative autoradiogram after 12% SDS-PAGE. Immunoprecipitates are from supernatants of mononuclear cells stimulated overnight with 1
mg/ml of LPS in the presence of [35S]methionine. From left to right are protein A-Sepharose immunoprecipitations with rabbit preimmune serum (pre), with
rabbit carboxyl terminus-specific IL-1b antiserum (Rc), and with rabbit amino terminus-specific IL-1b antiserum (Rn). It should be noted that four distinct
forms can be detected: a 31-kDa form by both Rc and Rn, a 28-kDa form by Rc only, a 17-kDa form by Rc only, and a 3-kDa form by Rn only. This is
typical of three separate immunoprecipitations.
The Journal of Immunology
4861
again demonstrating that capture by the B1 Ab-specific epitope is
impaired.
Binding of released proIL-1b to type II IL-1R. We have demonstrated recently that cytosolic proIL-1b can bind the type II
IL-1R (31). To determine whether released proIL-1b could also
bind the type II IL-1R, [35S]methionine-labeled cytosolic and released proIL-1b forms were incubated with recombinant type II
IL-1R and immunoprecipitated with a nonneutralizing mAb to the
type II IL-1R, M2. M2 complexes were retrieved by protein ASepharose beads, eluted into Laemmli sample buffer by boiling,
and analyzed by SDS-PAGE and autoradiography. Fig. 5 demonstrates that released mature IL-1b and cytosolic proIL-1b could be
immunoprecipitated by this procedure, but that released proIL-1b
could not. Furthermore, the released proIL-1b was not complexed
to another protein since denatured and nondenatured samples of
released proIL-1b ran identically to the recombinant proIL-1b
standard (data not shown).
Finally, to demonstrate that [35S]methionine labeling did not
interfere with the ability of released proIL-1b to bind the type II
receptor, an ELISA format was devised to test for released
Table I. Comparison of proIL-1b release by two ELISA formats a
ELISA Format (pg/ml)
Source
Monocyte
1
2
3
4
5
Macrophage
1
2
3
4
5
B1/Rn
Rn/Go
0
0
0
13
0
620
612
682
837
971
13
160
0
30
63
98
479
380
604
803
a
Normal human monocytes and alveolar macrophages (106/ml) from five different
individuals (1–5) were cultured overnight in RPMI 1640, 5% FBS, and 1 mg/ml LPS.
Supernatants were harvested and assayed for proIL-1b by two ELISA formats (B1/Rn
and Rn/Go, as described in Materials and Methods).
FIGURE 5. ProIL-1b immunoprecipitation via the type II IL-1R. The
ability of supernatant (Supn) and cytosolic (Cytos) form of proIL-1b to be
captured by the soluble type II IL-1R was used to compare the conformation of the two proIL-1b forms. Crude supernatant and cytosol fractions
from [35S]methionine-labeled PBMC after overnight stimulation with 1
mg/ml LPS were compared. Both fractions were incubated with (R1) or
without (R2) recombinant type II IL-1R at 10 ng/ml for 1 h at 20°C, and
then incubated with either IgG1 isotype control MOPC21 (MP21) or M2,
a nonneutralizing mouse monoclonal to the type II IL-1R. Immunoprecipitations were performed with protein G-Sepharose, and the protein bound to
beads was boiled into Laemmli sample buffer and analyzed by autoradiography of 12% SDS-PAGE. The cytosol panel (right) was exposed to film
overnight, while the supernatant panel (left) was exposed for 5 days.
proIL-1b binding to the type II IL-1R. In this format, functional
type II IL-1R are captured by the nonneutralizing M2 Ab, and
receptor-bound IL-1 can be detected with antisera specific to IL-1.
While [35S]methionine-labeled cytosolic proIL-1b and released
mature IL-1b complexed the immobilized type II IL-1R (as detected by Rn and Rc antisera, respectively), released proIL-1b did
not. These data imply that receptor binding sites in released
proIL-1b are altered significantly from those on cytosolic
proIL-1b.
Discussion
The processing and release of proIL-1b by mononuclear phagocytes are an important regulatory point for IL-1b. It has been long
recognized that abundant quantities of IL-1b remain intracellular
in stimulated monocytes and macrophages (10, 16). In some cell
types such as keratinocytes and to a lesser extent tissue macrophages, the predominant form of IL-1b after stimulation is intracellular proIL-1b (10, 16, 32). In this context, metabolic labeling
studies of newly synthesized IL-1b demonstrate that precursor
proIL-1b can be released intact from stimulated mononuclear
phagocytes. In the present study, utilizing both a specific proIL-1b
Ab and a traditional mature IL-1b-specific Ab, we show that IL-1b
is released in a number of forms other than 17 kDa. The prevalent
31-kDa form of released IL-1b is truly proIL-1b since immunoprecipitation by the proIL-1b-specific Ab is blocked by the immunizing peptide, while its immunoprecipitation by the mature
specific antisera is not. A 28-kDa form of released IL-1b is detected by the mature specific Ab, but not by the proIL-1b-specific
Ab. Importantly, a 3-kDa piece is also recognized by the proIL1b-specific antisera, but not by the mature specific antisera. The
3-kDa fragment detection is also inhibited by the 3–21 immunizing
peptide.
This is the first direct demonstration that ICE cleavage at Asp28
occurs during proIL-1b processing by monocytes and, furthermore, the first to show that this peptide is released from stimulated
mononuclear phagocytes. It confirms the suggestion by Higgins et
al. (28) that monocytes may clip proIL-1b at Asp28, as evidenced
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FIGURE 4. Western blot of released proIL-1b. After overnight culture
in the presence of 1 mg/ml LPS, normal human mononuclear cell supernatants and lysates were harvested as described in Materials and Methods
and analyzed after 12% SDS-PAGE and electrophoretic transfer to nylon
membranes. Shown is a representative blot from three different donors and
decreasing amounts of standard rproIL-1b. From left to right are rproIL1b, 1, 0.3, 0.1, and 0.03 ng/lane, followed by the lysate and supernatant,
respectively, from donors A, B, and C. Lysates represent 105 monocytes,
and supernatants represent 0.2 3 105 monocytes. By extrapolation to the
standard curve, the released proIL-1b is similar in amount to the remaining
intracellular proIL-1b.
RELEASED FORMS OF IL-1b
4862
undergoes a conformational change with processing, as evidenced
by proteinase K processing patterns (36). It was suggested that
cytosolic proIL-1b exists in a loosely folded conformation that
changes to a tightly folded conformation with processing. Applying this paradigm to the present data, one could postulate that the
change from loose to tight folding may happen as a consequence
of export from the cytosol. A change to a tight folding pattern
when the amino terminus remains intact could produce a molecule
whose receptor-binding epitopes are hidden. This would imply that
the conformational change is critical to export. It also suggests that
ICE processing is not required for this change. Knowledge of the
three-dimensional structure of proIL-1b will undoubtedly be helpful in understanding these events. The significance of the difference in the detectability of cytosolic vs released proIL-1b is unknown at present. The change has implications from a
measurement perspective and most likely is related to the poorly
understood processes that are necessary to release this molecule,
which lacks a leader sequence and is released via a non-Golgi
pathway.
These studies demonstrate that the release of IL-1b is not dependent on processing, that released proIL-1b is not readily detectable by capture techniques that recognize receptor binding, and
that with processing, precursor fragments of 28 and 3 kDa are also
produced. These studies suggest that a conformational change in
proIL-1b is part of the release process.
Acknowledgments
We thank Douglas Miller and Nancy Thornberry of Merck Research Labs
for providing the recombinant function ICE, and Steven Dower and
Michael Widmer of Immunex for the soluble IL-1 type II receptor and for
the Ab to the IL-1 type II receptor.
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by a 14-kDa band seen in their experiments. The significance of
this 3-kDa fragment is not known. However, the previous observation that the Asp28 processing site is not required for ICE to
cleave at the activation site of IL-1b, Asp116 (30), coupled with
our observation that this 3-kDa peptide is present extracellularly,
suggests the possibility of a unique function. Since the precursor
component of proIL-1b is highly conserved between IL-1a and
IL-1b, a functional role for the 3- and 11-kDa fragments should be
considered, as March et al. have previously suggested (18). It is
particularly noteworthy that 16-kDa N-terminal propiece of IL-1a
has been confirmed to be a nuclear oncoprotein (33). Although we
have not attempted to demonstrate function for the released 3-kDa
IL-1b propiece, the release of this molecule does have implications
for the relationship of ICE to the pathway. It implies that the release pathway is intimately associated with the processing machinery. It is consistent with the possibility that the ICE cleavage event
occurs on the outer membrane of monocytes and macrophages, as
has been suggested by the demonstration of constitutively active
ICE on monocyte plasma membranes by Singer et al. (34).
A recent observation has suggested that the ability to detect this
released form of proIL-1b is highly dependent upon the affinity of
the Abs used (28). In this context, we and others have confirmed
recently that intracellular concentrations of proIL-1b may be
greatly underestimated by ELISAs designed to detect mature
IL-1b (16, 35). However, in the process of analyzing our newly
described proIL-1b ELISA, we observed a unique deficiency in the
detection of released proIL-1b. The proIL-1b-specific ELISA that
readily detected cytosolic proIL-1b was virtually blind to the released forms of proIL-1b that could be detected by Western blotting or immunoprecipitation techniques.
To further characterize the released form of proIL-1b, a number
of experiments were performed to compare this form with the cytosolic form of proIL-1b. As mentioned, while the B1/Rn ELISA
format could not detect the released form of proIL-1b, it could
readily detect intracellular proIL-1b, as referenced to Western blot
detection (33). This inability to detect the released form of
proIL-1b was not due to degradation within the ELISA format
since [35S]methionine-labeled proIL-1b remained intact when captured and incubated in the ELISA system. Furthermore, a unique
ELISA format (Rn/G) was able to detect the released proIL-1b.
Since the B1 Ab blocks mature IL-1b function, but does not
recognize released proIL-1b, released proIL-1b was tested for its
ability to complex the type II IL-1R, another assay for the availability of functional epitopes. We have demonstrated recently that
cytosolic and recombinant proIL-1b can complex the type II receptor. However, in agreement with the masking of the B1 epitope,
released proIL-1b was also not detected by the type II IL-1R.
Attempts to explain the differences between these two forms of
proIL-1b to date have been unsuccessful. It is possible that the
released proIL-1b is part of a multimeric protein complex. However, in labeling studies, no [35S]methionine-labeled protein coprecipitates with the released proIL-1b. Likewise, attempts to immunoprecipitate released proIL-1b with antisera to ICE or the type
II IL-1R were also unsuccessful (data not shown). This does not
exclude the possibility that the released form of proIL-1b elutes
with constitutive proteins that are not labeled with methionine.
However, nondenaturing polyacrylamide electrophoresis with immunoblotting reveals released proIL-1b migrates identically with
the rproIL-1b standard. Thus, although it remains possible that the
released form of proIL-1b is part of a unique protein complex,
experiments to date do not support this explanation.
An alternative explanation is that proIL-1b is induced to undergo a conformational change in the process of secretion. Hazuda
et al. have demonstrated that the carboxyl terminus of proIL-1b
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