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and IL-1
, Function as an Antagonist and Agonist of NF-
B Activation Through the Orphan IL-1 Receptor-Related Protein 21
DNAX Research Institute of Molecular and Cellular Biology, Palo Alto, CA 94304
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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and IL-1
. Northern blot analyses show that
these IL-1s are highly abundant in embryonic tissue and tissues
containing epithelial cells (i.e., skin, lung, and stomach). In
extension, quantitative real-time PCR revealed that of human
skin-derived cells, only keratinocytes but not fibroblasts, endothelial
cells, or melanocytes express IL-1 and . Levels of keratinocyte
IL-1 are 
10-fold higher than those of IL-1. In vitro
stimulation of keratinocytes with IL-1
/TNF-
significantly
up-regulates the expression of IL-1 mRNA, and to a lesser extent of
IL-1 mRNA. In NF-
B-luciferase reporter assays, we demonstrated
that IL-1 and proteins do not initiate a functional response via
classical IL-1R pairs, which confer responsiveness to IL-1 and
or IL-18. However, IL-1 activates NF-B through the orphan
IL-1R-related protein 2 (IL-1Rrp2), whereas IL-1, which shows
striking homology to IL-1 receptor antagonist, specifically and
potently inhibits this IL-1 response. In lesional psoriasis skin,
characterized by chronic cutaneous inflammation, the mRNA expression of
both IL-1 ligands as well as IL-1Rrp2 are increased relative to normal
healthy skin. In total, IL-1 and and IL-1Rrp2 may constitute an
independent signaling system, analogous to IL-1/receptor agonist
and IL-1R1, that is present in epithelial barriers of our body and
takes part in local inflammatory responses. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, IL-1, and IL-1 receptor antagonist
(IL-1ra)3
(2, 3, 4), and surface and soluble IL-1 receptors (IL-1RI,
IL-1RII, and IL-1R accessory protein (5, 6, 7), termed
IL-1R1, IL-1R2, and IL-1R3 in this paper, respectively, in keeping with
our previously proposed numbering system (8). IL-1
signaling is initiated by high-affinity binding of IL-1 and to
IL-1R1, which gets subsequently bound by IL-1R3 (5, 7).
This results in an intracellular signaling cascade quite similar to
stress-induced signal transduction (9), with the end
effect being activation of NF-B (10). IL-1ra and IL-1R2
antagonize the response to IL-1 and at the ligand and
(co)receptor levels (2, 3, 11, 12). Numerous studies have
shown that perturbation of such control contributes to the pathogenesis
of inflammatory and immunological diseases (i.e., leukemia, rheumatoid
arthritis, and psoriasis; Refs. 13 and
14).
Recently, new members of the IL-1 family were identified based on both
sequence homology and the presence of key structural patterns. For
example, IL-18 (15, 16) is predicted to fold as a -rich
trefoil, typical for IL-1 ligands (17). Moreover, with
respect to processing, receptor usage, and signaling, IL-18 can be
classified as an IL-1 family member (i.e., IL-1
; Refs.
18, 19, 20, 21, 22, 23). In addition, new IL-1 ligands have been
identified that show strong structural similarities to IL-1ra
(24, 25, 26, 27, 28, 29). To date, the expression and especially the
receptor usage and function of these IL-1ra-like IL-1 ligands have only
been characterized to a limited extent. At the IL-1 receptor level,
there also exist additional IL-1R-like molecules. Many of these
molecules are currently orphan receptors, such as T1/ST2 (termed
IL-1R4; Refs. 30 and 31), IL-1R-related
proteins 1 (IL-1R5; Ref. 32) and 2 (IL-1R6; Ref.
33), IL-1R accessory protein ligand (IL-1R7; Ref.
18), single Ig domain IL-1R-related protein (IL-1R8; Ref.
34), IL-1R accessory protein-like (IL-1R9)
(35), and IL-1R10 (36), all harboring
extracellular Ig-folds and an intracellular domain homologous to the
cytosolic part of the Drosophila Toll protein. It is
interesting to note that the majority of the IL-1 ligands (i.e.,
IL-1/ra and some IL-1ra-like IL-1 ligands) and IL-1Rs (i.e.,
IL-1R1, IL-1R2, IL-1R4, IL-1R5, IL-1R6, and IL-1R7) are clustered and
localized to chromosome 2 (18, 25, 28, 37, 38, 39, 40).
The presence of several orphan IL-1Rs suggests the existence of
additional corresponding IL-1 ligands. In line with recent reports
(24, 25, 26, 27, 28), we have independently identified two novel IL-1
ligands based on sequence homology with IL-1ra, which we termed IL-1
and IL-1. IL-1 corresponds to the reported sequences of IL1Hy1
(24), FIL1 (25), murine IL1H3
(26), IL-1RP3 (27) and IL-1L
(28), whereas IL-1 corresponds to IL1H1
(26) and IL-1RP2 (27). These novel
IL-1s are strongly expressed in embryonic tissue and epithelial cells,
such as skin keratinocytes. The expression of IL-1, and to a lesser
extent of IL-1, is significantly up-regulated in
IL-1/TNF--stimulated human keratinocytes. Human IL-1 and
IL-1 proteins do not activate NF-B through the classical IL-1Rs,
i.e., the IL-1Rs used by IL-1 and (IL-1R1 and IL-1R3) or IL-18
(IL-1R5/IL-1R7). Instead, IL-1 activates this transcription factor
via IL-1R6, and this response is potently and specifically antagonized
by IL-1. Lesional psoriasis skin shows a substantially increased
expression of both the IL-1 ligands as well as their IL-1R. IL-1 and
and IL-1R6 probably constitute an independent signaling system,
present in epithelial barriers of our body, which may take part in
local inflammatory responses.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Recombinant human IL-1, IL-1, IL-4, IFN-, and TNF-
were provided by R&D Systems (Minneapolis, MN). Recombinant human IL-18
and IL-1ra were produced at DNAX Research Institute of Molecular and
Cellular Biology (Palo Alto, CA). The Q293 and 293-T cell lines were
maintained in DMEM supplemented with 5% FBS, 0.3 mg/ml
L-glutamine, 100 U/ml penicillin G, and 100 µg/ml
streptomycin (Life Technologies, Paisley, U.K.). Human primary
epidermal keratinocytes, dermal fibroblasts, dermal microvascular
endothelial cells, and melanocytes (Clonetics, San Diego, CA) were
cultured in specialized growth medium according to the suppliers
recommendations. The Jurkat E6.1 cell line was maintained in RPMI 1640
medium supplemented with 10% FBS, glutamine, and antibiotics.
Cloning of human and mouse IL-1 and IL-1
BLAST searches in the public mouse expressed sequence tag
(EST) database with the common portion of murine IL-1ra revealed EST
mb49b11.r1 (GenBank accession no. W08205). The insert contained the
full-length sequence of a novel IL-1-like molecule, designated IL-1.
With this mouse sequence as a query, a human EST (5120028H1), derived
from RNA from bronchial smooth muscle cells, was found in our
proprietary Incyte database (Palo Alto, CA) that contained the
full-length open reading frame of the human ortholog of mouse IL-1.
The same query sequence revealed an additional EST mi08c10.r1 in the
public mouse database (GenBank accession no. AA030324), which contained
partial sequence of a second novel IL-1-like molecule, designated
IL-1. The full-length sequence of murine IL-1 was obtained by
extending the 5' sequence by PCR on murine 17-day-old embryo
Marathon-Ready library cDNA (Clontech, Palo Alto, CA). Separately, a
Hidden Markov Model HMMer search (http://hmmer.wustl.edu/) with
a PFAM alignment of IL-1, IL-1, and IL-1ra
(http://pfam.wustl.edu/) revealed an EST (HAICR08) derived from
RNA from epithelial cells in the Human Genome Sciences database
(Rockville, MD) that contained the full-length open reading frame of
human IL-1.
A multiple alignment of these novel IL-1 sequences and published IL-1 sequences was created using CLUSTALW (41), guided by tertiary structures and predicted secondary structures (with a consensus derived from several algorithms at http://circinus.ebi.ac.uk:8081/submit.html), and refined by eye. Conserved alignment patterns were drawn by Consensus (http://www.bork.embl-heidelberg.de/Alignment/consensus.html). Evolutionary tree analysis was performed with a neighbor-joining algorithm and viewed with TreeView 1.5 (http://taxonomy.zoology.gla.ac.uk/rod/treeview.html).
Protein expression and purification of human IL-1 and IL-1
Adenoviral vectors containing full-length human IL-1 and
IL-1 sequences were constructed by PCR and used to transfect Q293
packaging cells. Viruses were subsequently purified, with all
procedures according to the manufacturer’s protocols (Invitrogen,
Carlsbad, CA). Q293 cells (5 x 108) were
infected (adenoviruses used at 10 multiplicity of infection) and
incubated for 5 days in a cell factory in a total volume of 1 L of
serum-free CMF-1 medium (Life Technologies). Culture medium was
dialyzed (Spectra/Por membrane tubing; molecular mass cut-off, 6–8
kDa; Spectrum Laboratories, Rancho Dominguez, CA) against 50 mM
Tris-HCl, pH 8.0, and 1 mM EDTA, and subsequently passed through hitrap
Q Sepharose and heparin columns. The flow-through, containing the IL-1
proteins, was sterile-filtered and concentrated 70 times with an
Amicon 8400 ultrafiltration cell with a 10-kDa molecular mass cut-off
membrane (Millipore, Bedford, MA). The samples were dialyzed against
PBS, and the protein content was quantified by PAGE and Coomassie blue
staining with lysozyme as a standard. Protein identities were confirmed
by N-terminal sequencing. Identically treated culture medium of Q293
cells infected with adenovirus encoding green fluorescent protein
served as a negative control. Endotoxin levels were determined by using
the Limulus amebocyte lysate assay (BioWhittaker,
Walkersville, MD) and were <1.5 EU/100 µg protein. Protein samples
were stored at 4°C.
Expression plasmids
Plasmids encoding full-length human R1, mouse R3, mouse R4,
human R5, and human R7 sequences were constructed by inserting
PCR-generated cDNA fragments into pME18S (42). Human
IL-1R6 cDNA, a generous gift of Dr. R.A. Maki (Neurocrine Biosciences,
San Diego, CA), was subcloned directly into pME18S. The reporter gene
plasmid pNF-B-Luc (Stratagene, La Jolla, CA) contains five NF-B
sites and a basic promoter element to drive luciferase expression, and
pRSV-Gal results in constitutive expression of
-galactosidase.
Northern blots
Mouse Northern blots containing 2 µg of
poly(A)+RNA per lane, derived from either total
embryo at different days postgestation (Clontech) or from various adult
tissues (Origene Technologies, Rockville, MD), were hybridized to the
mouse IL-1 and IL-1 cDNA probes containing the complete open
reading frames. Probes were labeled with 32P by
using the Redivue labeling kit (Amersham Pharmacia Biotech, Uppsala,
Sweden). Prehybridization, hybridization, stringency washes, and
stripping were conducted according to the manufacturer‘s protocols.
Membranes were exposed to a phosphorimager.
Quantitative real-time PCR
A panel of various human skin-derived cells, i.e., primary
epidermal keratinocytes, dermal fibroblasts, dermal microvascular
endothelial cells, and melanocytes, as well as skin biopsies and PBMC
were used for TaqMan-PCR analyses. Skin-derived cells were left
untreated or were treated with IL-4 (50 ng/ml), IFN- (20 ng/ml), or
a combination of IL-1 (5 ng/ml) and TNF- (10 ng/ml) for 18 h
before RNA isolation. Biopsies from lesional psoriasis skin and normal
healthy skin were kindly donated by Dr. T. Ruzicka (Department of
Dermatology, University of Dusseldorf, Germany) and homogenized before
RNA isolation. PBMC from a healthy donor, prepared by standard
protocols, stimulated with and without PHA served as controls. RNA
isolation, cDNA synthesis, and PCR were performed as described
elsewhere (43). The amplicons for human IL-1 (nt
17–90, with numbers starting at first methionine codon), human IL-1
(nt 337–409), and human IL-1R6 (nt 1378–1448) were analyzed with
6-carboxy-fluorescein-labeled probes. 18S RNA quantities were
measured with a VIC-labeled probe and served as internal controls to
normalize for the total amount of cDNA. Values are expressed as fg/5 ng
total cDNA.
Reporter assay
Jurkat E6.1 cells (4 x 106) were
transiently transfected with pNF-B-Luc reporter gene plasmid,
pRSV-Gal plasmid, and IL-1R-encoding cDNA plasmid(s) as described
previously (43). Twenty hours after transfection, cells
were stimulated with 20 ng/ml of human IL-1, IL-1, IL-18, or
IL-1ra, or 50 ng/ml human IL-1 or IL-1 for 6 h. Cells were
lysed with reporter lysis buffer (Promega, Madison, WI), and luciferase
and -galactosidase activities were assessed with luciferase assay
reagent (Promega) and Galacto-Light Kit (Tropix, Bedford, MA),
respectively. Luciferase activities (in RLU) were normalized on the
basis of -galactosidase activities. For inhibition studies of
IL-1R1-mediated activation of NF-B, IL-1 was used at 50 pg/ml in
the presence of IL-1ra and IL-1 at concentrations ranging from 10
pg/ml to 10 µg/ml. Inhibition of the IL-1R6-mediated response was
analyzed with IL-1 used at 50 ng/ml and IL-1 or IL-1ra at
concentrations ranging from 64 pg/ml to 10 µg/ml.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and were identified computationally
Computational analyses led to the discovery of two novel IL-1
ligands: IL-1 and IL-1. In short, the strategies used both
homology-based and probabilistic-based (HMMer) searches (see
Materials and Methods for details). The sequences, and a
comparison with the previously known IL-1 family members, are given in
Fig. 1
A. Fig. 1B,
the corresponding dendrogram, shows evolutionary relationships.
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and IL-1 messenger RNA are highly expressed in embryonic
tissue and in epithelial cells
Northern blot analyses show that IL-1 and IL-1 are expressed
in embryonic tissue and tissues containing epithelial cells (i.e.,
stomach and skin; Fig. 2). Quantitative
PCR analyses on a large panel of mouse and human tissue cDNAs
(including various lymphoid organs, kidney, heart, lung, brain, liver,
organs of the digestive tract, reproductive organs, and skin) confirmed
these findings. Messenger RNA expression in lung tissue appears to be
unique to IL-1 (Fig. 2). It should also be noted that IL-1 mRNA
analysis shows the presence of multiple variants. More in-depth
studies, based on quantitative PCR, revealed that in skin,
keratinocytes but not fibroblasts, endothelial cells, or melanocytes
are the main producers of IL-1 and IL- (Fig. 3). In vitro-cultured keratinocytes
contained 10-fold more IL-1 mRNA relative to IL-1 mRNA.
Stimulation with IL-4 or IFN- hardly affected the expression levels
of IL-1 and mRNA, whereas stimulation with a combination of
IL-1 and TNF- resulted in an enormous increase in the expression
of IL-1 mRNA and to a lesser extent of IL-1 mRNA (Fig. 3).
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and IL-1 do not activate NF-B through classical
IL-1Rs
The ability of the novel IL-1s to initiate IL-1R-mediated
signaling was studied via an NF-B-dependent reporter assay with
ligand-stimulated Jurkat T cells transiently transfected with different
pairs of IL-1Rs. The R1/R3 combination, conferring responsiveness to
IL-1 and IL-1 (5, 44), did not generate a response
to IL-1 or IL-1. Also, the R5/R7 combination, required to mediate
a response to IL-18 (18, 43), did not result in signaling
on addition of the novel IL-1 ligands (Fig. 4A). The next step was to test
the orphan IL-1R-like molecules IL-1R4 and IL-1R6 (classified as
potential ligand-binding receptors based on their homology to IL-1R1;
Ref. 36) paired with various other IL-1R-like molecules,
i.e., IL-1R3, IL-1R7, IL-1R9, and IL-1R10 (classified as potential
signaling receptors based on their homology to IL-1R3; Ref.
(36) for their capacity to confer responsiveness to
IL-1 and IL-1. Data consistently showed an IL-1R6-mediated
activation of NF-B upon stimulation with IL-1, but not IL-1 or
the mock control (Fig. 4B).
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specifically and very potently antagonizes the IL-1R6
response to IL-1
In line with the striking similarity between IL-1 and IL-1ra,
we tested the possibility of IL-1 being an antagonist of IL-1
responses rather than being an IL-1 agonist. With the same reporter
assay with IL-1R-transfected Jurkat T cells, we showed that IL-1ra, but
not IL-1, is able to antagonize the IL-1R1-mediated activation of
NF-B on stimulation with IL-1 (Fig. 5A). Vice versa, IL-1, but
not IL-1ra, is able to antagonize the IL-1R6-mediated activation of
NF-B on stimulation with IL-1 (Fig. 5B). Importantly,
IL-1ra shows a 50% inhibition of the IL-1R1-mediated response to
IL-1 at about a 1000-fold excess over IL-1, whereas IL-1
results in a similar inhibition of the IL-1R6-mediated response to
IL-1 at concentrations similar to or even less than IL-1.
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and and IL-1R6 levels are substantially up-regulated in
lesional psoriasis skin
In lesional psoriasis skin, characterized by chronic cutaneous
inflammation, the expression of the novel IL-1 ligands, IL-1 and
IL-1, and IL-1R6 are all significantly increased relative to
skin from a healthy individual (Fig. 6). The increase is most prominent for
IL-1, in line with in vitro-cultured keratinocytes stimulated with
the pro-inflammatory cytokines IL-1/TNF- (Fig. 3
). Activation of
PBMC also leads to increased levels of both IL-1 ligands and their
receptor, albeit to a lesser extent than observed in lesional
psoriasis skin.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and IL-1. Several recent studies have
reported on the cloning and molecular characterization of IL-1 and
IL-1 (24, 25, 26, 27, 28). However, the present study is the first
to report on the expression in human skin-derived cell types, receptor
usage, and initial functional characterization of IL-1 and
IL-1.
The structurally aided alignment of both the novel and classical IL-1s
in Fig. 1A shows the conservation of the core 12
-strands, making up the -trefoil structure. The presented
sequence of human IL-1 protein is identical with the reported
sequences of IL1Hy1 (24), FIL1 (25),
IL-1RP3 (27), and IL-1L (28). At the amino
acid level, human IL-1 and IL-1ra show a high degree of similarity,
which is confirmed by the evolutionary tree analysis (Fig. 1B). With respect to IL-1, the presented sequence is
identical with the sequences of IL1H1 (26) and IL-1RP2
(27). It is interesting to note that several public mouse
ESTs exist, mostly derived from tongue epithelium, with only slight
variations relative to the IL-1 sequence. In addition, FIL1
(25) also shows a very high similarity to the human
IL-1 sequence presented in this paper (i.e., 51%). How these
IL-1 variants are generated and their biological significance remain
unclear.
IL-1 and IL-1 are strongly expressed in embryonic development and
in tissues such as stomach, lung, and skin (Fig. 2 and PCR analyses on
a panel of various tissue cDNAs not shown). Lung tissue only showed
expression of IL-1 messenger RNA (at the Northern blot level),
although at the PCR level, expression of both IL-1 and IL-1 can
be detected in lung-derived cDNAs. Sizes of the predominant messages
for IL-1 are 1.4 and 2.7 kb for stomach and skin tissues,
respectively, and 2.0 kb for lung tissue. Interestingly, IL-1
messenger RNA in lung tissue is reported to lack the second exon
relative to other tissues (25). In analogy to IL-1ra, the
different IL-1 messages might reflect different (tissue-specific)
splice variants (2, 45, 46, 47). In fact, both IL-1 and
IL-1ra mRNA sequences diverge at the 5' ends because of usage of
alternative first exons (27). A detailed analysis of human
skin was performed with quantitative PCR analysis on a panel of
first-strand cDNAs derived from various skin-specific cell types.
Keratinocytes, but not fibroblasts, endothelial cells, or melanocytes
were identified as the major source for IL-1 and IL-1, with
levels of IL-1 being 10-fold higher than those of IL-1 (Fig. 3). In addition, Langerhans cells but not skin-homing T cells, freshly
isolated from skin biopsies, showed some expression of IL-1 and
(data not shown). In vitro stimulation of keratinocytes with
proinflammatory cytokines (i.e., IL-1/TNF-) but not with IL-4 or
IFN- significantly up-regulated the expression of IL-1, and to a
lesser extent of IL-1 (Fig. 3). Our observations are in line with
reports on the constitutive and induced expression of keratinocyte
IL-1 and IL-1 mRNA (26, 27). Preliminary in situ
hybridization data using mouse tissue sections confirmed that cells of
epithelial origin, such as the parietal and chief cells in stomach, and
basal keratinocytes in skin are the predominant cellular sources of
these IL-1s (not shown). In addition, esophageal squamous epithelium is
also reported to express IL-1 (27). The presence of
IL-1 in epithelial barriers of our body (i.e., skin, digestive,
and respiratory tracts), suggests that these novel IL-1s fulfill
similar roles as their known family members (i.e., IL-1 and IL-1)
to promote a response to injury or infection (1, 48). In
fact, Kumar and colleagues have shown that the epidermal expression of
murine IL-1 is up-regulated in vivo in response to contact
hypersensitivity or a viral infection (26).
It is important to note that IL-1 and IL-1 neither possess a
classical leader sequence (as does secreted IL-1ra; Ref.
2) nor do they possess a distinct pro-form (as do
IL-1 and IL-18; Refs. 4, 15 , and 16).
However, monitoring the presence of C-terminally tagged versions of
IL-1 and IL-1 in the supernatants and lysates of transfected
293-T cells (human epithelial cells) revealed that these molecules are
secreted as 20-kDa proteins (data not shown). This is in agreement with
the finding that the human trophoblastic tumor cell line JEG-3 is able
to secrete IL-1 (28), and argues that an alternative
mechanism exists to secrete these novel IL-1s. To functionally
characterize the novel IL-1s, we expressed and purified adenovirally
derived human IL-1 and IL-1 and tested these proteins for their
capacity to initiate IL-1 signaling, with NF-B activation as a
read-out. The observation that IL-1R1/3 and IL-1R5/7 do not respond to
these new protein preparations (Fig. 4A) might be explained
by the fact that receptor-ligand combinations within the IL-1 system
are very specific (43). Therefore, we subsequently tested
the orphan receptors IL-1R4 and IL- 1R6 paired with various other
IL-1R-like molecules. These studies consistently showed that IL-1R6
responded to IL-1 but not IL-1 in activating NF-B in Jurkat
cells (Fig. 4B). Even IL-1R6 single transfectants showed
this response. The IL-1 system, as we know it today, typically requires
two receptors, a ligand-binding subunit and a signaling subunit, to get
an IL-1 response (5, 18). Because IL-1R6 is very
homologous to IL-1R1 (33), a ligand-binding type of
receptor, we believe that Jurkat cells endogenously express a second
signaling type of receptor that can pair with IL-1R6 in the presence of
IL-1. We know that the following IL-1R-like molecules are expressed
by nontransfected Jurkat cells: IL-1R3, IL-1R4, IL-1R8, IL-1R9, and
IL-1R10 (PCR data, not shown). Cotransfection of IL-1R6 with either
IL-1R3, IL-1R9, or IL-1R10 does not potentiate the response to IL-1
relative to IL-1R6 single transfectants (not shown). In addition,
studies by others with IL-1R1 chimeras and IL-1-mediated activation
of NF-B as a read-out do not support a combination of IL-1R6 and
IL-1R8 to mediate an IL-1 response (34). The search for
the additional IL-1 receptor(s) is currently ongoing.
IL-1 is most closely related to IL-1ra, and, like IL-1ra, lacks the
loop between the fourth and fifth -strands (see Fig. 1A),
which is typical for IL-1 agonists: IL-1, IL-1, IL-18 and
IL-1. In fact, insertion of the loop amino acids QGEESN of IL-1
confers agonist activity to IL-1ra (49). Therefore, we
hypothesized that IL-1 acts as an antagonist. Indeed, IL-1 is a
very potent antagonist of the IL-1R6-mediated response to IL-1 at a
ratio of IL-1:IL-1 <1 (Fig. 5). Note that the potency of IL-1ra
to antagonize the IL-1R1-mediated response to IL-1 is 3 orders of
magnitude less. The observation by others (25) that their
FIL1 and FIL1 proteins do not bind to IL-1R6 is in our opinion
not contradictory to our findings. Binding studies with partially
purified IL-1 and IL-1 proteins from conditioned medium of
transfected cells and an Fc fusion of IL-1R6 might not be sensitive
enough to show binding to these new IL-1s. Moreover, the second
receptor might actually be needed for affinity conversion and for
binding to become detectable (5, 43).
IL-1 is a highly specific antagonist of the IL-1R6-mediated response
to IL-1. For instance, IL-1 does not respond through IL-1R1,
either as an agonist or antagonist (see Figs. 4 and 5), which confirms
the reported lack of IL-1 to induce the production of IL-6 or
inhibit the IL-1-induced production of IL-6 by cultured
fibroblasts or endothelial cells (28). Moreover, IL-1
does not respond through IL-1R5 because IL-1 does not induce the
production of IFN- or inhibit the IL-18-induced production of
IFN- by KG-1 cells (28). In fact, a recently cloned
IL-1ra homologue, termed IL-1H (with various isoforms: FIL1
(25), IL1H4 (26), and IL-1RP1
(27)) was shown to bind to IL-1R5 but not IL-1R1
(29), and may act as a specific IL-18 antagonist.
Expression of human IL-1R6 is restricted to lung epithelium and brain
vasculature (33). In extension to these findings, we
observed expression of IL-1R6 mRNA in monocytes and in skin-derived
keratinocytes, fibroblasts and to a lesser extent endothelial cells.
With respect to skin cells, IL-1R6 may in fact mediate proliferation
and production of matrix metalloproteinases in response to IL-1
(preliminary data, not shown). Activated monocytes also show an
up-regulated expression of IL-1 and IL-1 mRNA that probably
explains the presence of these IL-1s in activated PBMC (Fig. 6). The
expression of IL-1 and IL-1, as well as IL-1R6, mRNA are all, but
most notably IL-1 mRNA, highly increased in lesional psoriasis skin
samples relative to normal control skin samples (Fig. 6). These data
are momentarily followed up, but already confirm the involvement of
these novel IL-1s in response to skin inflammation (26)
and extend the notion that IL-1 ligands and receptors contribute to the
pathogenesis of psoriasis (13).
Taken together, IL-1 and and IL-1R6 may constitute an
independent signaling system analogous to IL-1/ra and IL-1R1. The
IL-1R6 system, present in epithelial barriers of our body, as a result
from the coexpression of IL-1 and IL-1, may be in a default
off-state. However, perturbation of homeostasis can shift this balance
to an IL-1-mediated inflammatory or proliferative response, as seen
in lesional psoriatic skin.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Robert A. Kastelein, DNAX Research Institute of Molecular and Cellular Biology, 901 California Avenue, Palo Alto, CA 94304. E-mail address: Kastelein{at}dnax.org
3 Abbreviations used in this paper: IL-1ra, IL-1 receptor antagonist; EST, expressed sequence tag.
Received for publication March 2, 2001. Accepted for publication June 1, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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