Received for publication, September 11, 2000, and in revised form, October 25, 2000
We report identification of interleukin (IL)-17E,
a novel member of the IL-17 family of cytokines. IL-17E is a ligand for the recently identified protein termed EVI27/IL-17BR, which we term
IL-17 receptor homolog 1 (IL-17Rh1) in light of the multiple reported
ligand-receptor relationships. Murine EVI27 was identified through its
location at a common site of retroviral integration in BXH2 murine
myeloid leukemias. IL-17Rh1 shows highest level expression in kidney
with moderate expression in multiple other organs, whereas IL-17E
mRNA was detected at very low levels in several peripheral tissues.
IL-17E induces activation of NF-
B and stimulates production of the
proinflammatory chemokine IL-8.
 |
INTRODUCTION |
IL-171 was identified as
a cellular ortholog of a protein encoded by the T lymphotropic
herpesvirus Saimiri (1-3). Subsequent characterization has
shown that this protein is a potent cytokine that acts to induce
proinflammatory responses in a wide variety of peripheral tissues
(reviewed in Ref. 4). Recently, two new proteins were identified termed
IL-17B and IL-17C that are clearly related to IL-17, establishing that
there exists a family of IL-17-like molecules (5). Initial
characterization of these proteins suggests that they also are able to
induce production of certain proinflammatory cytokines, suggesting that
a proinflammatory response may be a common feature of this family of
cytokines. Interestingly, they do not appear to be ligands for the
IL-17 receptor, suggesting that there exists other molecules that serve
as cognate receptors for these factors.
Interest in this family of molecules has increased as it has become
apparent that IL-17 may contribute to a number of important medical
conditions. In particular, several studies have pointed to a
relationship between IL-17 and rheumatoid arthritis (6-10). IL-17 may
also play a role in transplant rejection (11-13), psoriasis (14),
multiple sclerosis (15), and promotion of tumor growth (16). Given the
potential of IL-17-related molecules to occupy important roles in the
control of immune function, there is interest in the identification of
other members of this family and the receptors that direct the actions
of these molecules through particular target cell populations. In this
report we identify a novel member of this family and its receptor.
 |
EXPERIMENTAL PROCEDURES |
Isolation of IL-17E and Construction of Expression
Vectors--
IL-17E (DNA147531) and IL-17Rh1 (DNA115291) cDNA
clones were isolated from a human cDNA library and sequenced in
their entirety. Fc fusion proteins (immunoadhesins) were prepared by
fusion of the entire open reading frames of IL-17B, -C, and E in frame
with the Fc region of human IgG1 in the eukaryotic expression vector pRK5tkNEO and the baculovirus vector pHIF, a derivative of pVL1393 purchased from Pharmingen. Fusion proteins were transiently expressed in human 293 cells or Sf9 insect cells and purified over a
protein A column. The extracellular domain of IL-17Rh1 was also
expressed as a C-terminal 8×His tag fusion in baculovirus and purified
by nickel affinity column. IL-17E was also expressed as a 8×His tag fusion in Escherichia coli and was purified and refolded.
The identities of the purified proteins were verified by N-terminal sequence analysis.
Western Blot, Northern Blot, and Taqman
Analysis--
Western blot analysis of binding of IL-17E to IL-17Rh1
was performed essentially as described previously (17, 18). For Northern blot analysis, multiple tissue Northern blots
(CLONTECH) were probed with a
32P-labeled probe of random-primed IL-17Rh1 cDNA
according to manufacturer's recommendations and exposed to Kodak
X-omat (Eastman Kodak Co.) for 72 h. For quantitative PCR analysis
(Taqman), total mRNA from human tissues (50 ng) was analyzed
as recommended (PerkinElmer Life Sciences) with primers based on the
coding sequence of IL-17Rh1.
FACS Analysis--
Human 293 cells were transiently
cotransfected with expression vectors for green fluorescent protein
(GFP) and IL-17Rh1 or IL-17R as indicated. After 24 h, cells were
incubated with Fc-tagged ligand as indicated, and binding was revealed
with PE-conjugated anti-human Fc antibody. FACS curves show PE staining
within the cotransfected GFP-positive cell population.
NF-
B Assays, IL-8 Assays, and Western Blot
Analysis--
Luciferase reporter assays were conducted essentially as
described (19). Briefly, 293 or TK-10 cells (2 × 105)
were transfected by Effectine (Qiagen) transfection with 0.5 µg of
the firefly luciferase reporter plasmid pGL3-ELAM.tk and 0.05 µg of
the Renilla luciferase reporter plasmid as internal transfection control as well as IL-17E expression plasmid (0.1 µg)
and carrier plasmid pRK5D to maintain constant DNA between transfections. After 24 h cells were harvested and luciferase activity was assayed as recommended (Amersham Pharmacia Biotech). IL-8
enzyme-linked immunosorbent assay was performed according to
manufacturer's instruction (R&D Systems).
 |
RESULTS |
Analysis of human genomic DNA sequence information available in
GenBankTM (accession number CNS01DTR) led us to the
identification of a putative new member of the IL-17 family. A
full-length cDNA was obtained that corresponded to this gene (Fig.
1A). The encoded protein is
177 amino acids and is 16-20% identical to IL-17, IL-17B, and IL-17C.
We termed this new member of this expanding family IL-17E. The four
members share greatest similarity in the C-terminal portion of the
molecule with 20-30% amino acid identity and strict conservation of
four cysteines. Additional cysteines that may be functionally conserved
are present with differences in position. In contrast, there is little
conservation apparent in the N-terminal 80 residues. IL-17E mRNA
was not detected by Northern blot analysis. However, IL-17E was
detected at very low levels in several tissues including brain, kidney,
lung, prostate, testis, spinal cord, adrenal gland, and trachea by
reverse transcription-PCR using primers designed to distinguish spliced
mRNA from genomic DNA (Fig. 1B).

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Fig. 1.
Sequence and mRNA expression of
IL-17E. A, shown is alignment of the IL-17 family
members. The predicted signal sequences are underlined.
Conserved cysteines are indicated by bullets, and potential
N-linked glycosylation sites are boxed. B,
reverse transcription-PCR analysis of IL-17E expression. RNA from the
indicated tissues was subjected to reverse transcription-PCR with
primers that designed to amplify the entire coding sequence of IL-17E.
PCR product was resolved by agarose gel electrophoresis, transferred to
nylon membrane, and probed with a 32P-labeled IL-17E
cDNA probe.
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Previous work demonstrated that IL-17B and IL-17C do not bind IL-17R,
suggesting that there likely exist additional receptors that serve as
cognate pairs for members of this family of cytokines. To identify
candidate receptors, expressed sequence tags were examined for
sequences related to IL-17R. On the basis of one such group of
expressed sequence tags, a cDNA was isolated that encoded a
502-amino acid single transmembrane protein that shares 26% amino acid
identity to IL-17R. This molecule appears to be the same gene recently
reported as a receptor for IL-17B (20) and independently as EVI27, a
cDNA up-regulated by retroviral integration in BXH2 murine myeloid
leukemias (21). In light of our data described below that this is also
a receptor for IL-17E, we propose that this new receptor be termed
IL-17Rh1, a flexible nomenclature to reflect the possibility of
additional ligand-receptor relationships within this expanding receptor
ligand family. Our cDNAs encoded a protein with a substantially
longer C-terminal intracellular domain than that reported by Shi
et al. (20) (Close examination of their cDNA
reveals the presence of a frameshift relative to our clones.)
Our sequence agrees with the sequence reported by Tian et
al. (21) with one amino difference (a leucine versus
phenylalanine polymorphism at position 468) and shows close homology across full-length of the encoded protein with the murine ortholog, suggesting that this is likely the bona fide
sequence. IL-17Rh1 mRNA expression was examined by Northern blot
analysis (Fig. 2A) and
quantitative PCR (Fig. 2B). Highest levels of expression were observed in kidney with significant expression also observed in
liver and other peripheral organs.
To determine whether this new molecule serves as a receptor for members
of the IL-17 family, binding studies were conducted. Human 293 kidney
cells transfected with an expression vector for IL-17Rh1 bind IL-17E-fc
fusion protein (immunoadhesin) but do not show significant binding of
IL-17 (Fig. 3A). IL-17E
immunoadhesin binding to IL-17Rh1 expressing cells could be completely
inhibited by competition with His epitope-tagged IL-17E (not shown). In comparison, cells transfected with expression vector for IL-17R bind
IL-17 immunoadhesin but not IL-17E. To examine whether there was direct
interaction with members of the IL-17 family, ligand binding studies
were conducted with epitope-tagged extracellular domain of IL-17Rh1. We
find that this new molecule exhibits robust binding of IL-17E-fc and
weak binding to IL-17B-fc but does not bind IL-17-fc or IL-17C-fc (Fig.
3B).
IL-17Rh1 is the second receptor identified that binds to members
of the IL-17 family. The IL-17 receptor family is quite unrelated to
other known proteins. The proteins do not possess any recognizable protein structural domains. However, comparison of the two receptors does reveal conservation of many cysteines within the extracellular domain, suggesting that they share similar structure. There are conserved elements within the intracellular domain as well, suggesting that these receptors likely engage similar intracellular machinery. This is supported by the observation that like IL-17 IL-17E signals activation of NF-
B. The regions of conservation within the
intracellular domain do not bear obvious similarity to two other
receptor families known to activate NF-
B, the IL-1/Toll and TNF
receptor families. Details of IL-17 signaling pathways have not yet
been well established although numerous intracellular events have been
reported including activation of STATs (22), Raf-1 kinase (23), and
several mitogen-activated protein kinases (9). The recent demonstration
that IL-17 signaling is deficient in TRAF6-deficient cells strongly
suggests that members of the TRAF family, known to be involved in both
IL-1/Toll and TNF receptor signaling, are also involved in IL-17
receptor signaling (24).
IL-17E induces production of IL-8, a proinflammatory molecule that has
also been observed to be induced by IL-17, suggesting that the
biological activities of these two cytokines may be similar. The IL-17
receptor has a very broad expression pattern in contrast to the
somewhat more restricted mRNA expression pattern of IL-17Rh1. If
these molecules mediate generally analogous proinflammatory responses,
a key consideration in understanding the function of the different
members of the expanding IL-17 cytokine family will be the expression
patterns and regulation of the cognate receptors. The observation
that IL-17Rh1 may bind two distinct ligands has parallels in other
receptor systems that activate NF-
B. Both the IL-1 family and the
TNF receptor superfamily have numerous examples of promiscuous
interactions between ligands and receptors (reviewed in Refs.
25-28).
The murine ortholog of this new receptor was identified at Evi27, a
common site for retroviral integration in BXH2 murine myeloid
leukemias. The human IL-17Rh1 maps to 3p21, a region that has been
noted to undergo deletion in a variety of cancers, notably renal cell
carcinoma (29-32). Interestingly, this region is also a frequently
deleted region in chronic myelogenous leukemia (33). A number of other
cytokine and growth factor receptors have been implicated as oncogenes,
reflecting the important roles these proteins play in control of cell
growth and regulation of immune function. Aicardi-Goutieres syndrome,
an early onset progressive encephalopathy that may present with raised
white cell counts or raised levels of interferon-
in the
cerebrospinal fluid, also maps to this region (34). It will be of
interest to carefully examine the role this cytokine signaling system
may play in human disease, particularly cancer and chronic inflammatory conditions.
We thank Sherman Fong and Paul Godowski for
helpful discussions and Amy Carlow, Jeffrey Hooly, Peter Ng, and Mark
Vasser for technical assistance.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF305200.
Published, JBC Papers in Press, October 31, 2000, DOI 10.1074/jbc.M008289200
The abbreviations used are:
IL, interleukin;
Rh, receptor homolog;
PCR, polymerase chain reaction;
FACS, fluorescence-activated cell sorter;
GFP, green fluorescent protein;
PE, phosphatidylethanolamine;
STAT, signal transducer and activator of
transcription;
TNF, tumor necrosis factor.
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