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Department of Immunology, Geneva Biomedical Research Institute, Glaxo Wellcome Research and Development, Plan-les-Ouates, Switzerland
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-chain, or a subunit of a multimeric cytokine, e.g., IL-12 p40. The
highest levels of hCLF-1 mRNA were observed in lymph node, spleen,
thymus, appendix, placenta, stomach, bone marrow, and fetal lung, with
constitutive expression of CLF-1 mRNA detected in a human kidney
fibroblastic cell line. In fibroblast primary cell cultures, CLF-1 mRNA
was up-regulated by TNF-, IL-6, and IFN-
. Western blot analysis
of recombinant forms of hCLF-1 showed that the protein has the tendency
to form covalently linked di- and tetramers. These results suggest that
CLF-1 is a novel soluble cytokine receptor subunit or part of a novel
cytokine complex, possibly playing a regulatory role in the immune
system and during fetal development. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The receptors for the IL-6-type cytokines (10), including IL-6, ciliary neurotrophic factor (CNTF),5 IL-11, leukemia inhibitory factor (LIF), oncostatin M, and cardiotropin-1 are typical of the cytokine type I receptor family, forming multicomponent complexes comprising ligand binding and shared signaling subunits (11). Pleiotrophy and redundancy within this family can be accounted for by the fact that the receptors for many of these cytokines use the signaling subunits gp130 (12) or the LIF receptor (13), both with long intracytoplasmic tails and involved in the induction of the JAK-STAT signaling pathway (14, 15). Receptor specificity is provided by the ligand binding subunit. In the case of the IL-6, IL-11, and CNTF receptors (IL-6R, IL-11R, and CNTFR), a soluble form of this subunit is capable of rendering cells sensitive to the appropriate ligand provided the correct signaling subunit (gp130 or LIF receptor) is present on the cell surface (16, 17, 18, 19). In the case of the receptors for LIF and cardiotropin-1, LIF receptor serves as both a ligand binding and a signaling subunit (13, 20, 21), as is the case for gp130 within the oncostatin M receptor complex (22, 23).
Another interesting configuration is that demonstrated by the organization of IL-12 and its receptor. IL-12 has been shown to be composed of two covalently linked polypeptide chains known as p35 and p40. Certain regions of p35 share homology with IL-6 and granulocyte CSF, while p40 shares homology with the extracellular region of IL-6R and CNTFR (24). p40 also displays the hallmarks of the cytokine type I receptor family, containing the four conserved cysteines residues and a WSEWAS sequence, resembling the W-S-x-W-S motif (5). This has led to the suggestion that IL-12 may have originated from a cytokine/soluble cytokine receptor complex, a suggestion supported by the homology between the IL-12R and gp130 (25).
In this report, bioinformatics was used to identify human and murine homologues of a novel cytokine-like factor, CLF-1, which was subsequently cloned and analyzed. Human and murine CLF-1 share 96% amino acid identity, suggesting an important function for the protein. CLF-1 shares significant homology with many members of the cytokine type I receptor family. Analysis of CLF-1 mRNA distribution and recombinant forms of the CLF-1 protein provide the initial characterization for this novel member of the cytokine type I receptor family.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The amino acid sequence N K L C F D D N K L W S D W S E A Q S I G K E Q N from the murine IL-13R (26) was used to search the GenBank database with expressed sequence tags (ESTs) using TBLASTN to identify cDNAs encoding receptors with related sequence. BLASTN searches of the GenBank database with the identified EST allowed the identification of ESTs with overlapping sequence homology.
The mouse cDNA clone 479043 was purchased from Research Genetics (Birmingham, AL). 5'-RACE on poly(A)+ RNA extracted from mouse lung allowed the cloning of 310 bp of the murine cDNA upstream of the 5' end of the cDNA clone using the Marathon cDNA amplification kit (Clontech, Palo Alto, CA) following the manufacturer’s guidelines. The primer used in the PCR amplification (along with the adaptor protein-1 primer provided) was 5'-CGTACCACCTCAGCTTGTACTTG-3'. PCR products were cloned into the vector pCRII (Invitrogen, Leek, The Netherlands), and colonies were screened by hybridization with the oligonucleotide probe 5'-AAGGATCTCACGTGCCGCTGGACACCGGGT-3'.
A portion of the hCLF-1 cDNA was amplified by PCR using cDNA derived from human lung poly(A)+ RNA with the primers 5'-ACCGCCGAGGGCCTCTACTG-3' and 5'TTGAGGGAGTAGTTGGTGTGGAGG-3'. The amplified product was cloned into the vector pCRII and subsequently used as a 32P-labeled probe to screen a human placental cDNA library in gt10. The largest cDNA identified (1740 bp) was recloned in pBluescript II SK- (Stratagene, La Jolla, CA).
DNA and protein sequence analysis
Sequences obtained from cDNA clones as well as all relevant ESTs were imported into and analyzed by the sequence analysis software Sequencher (Genecodes, Ann Arbor, MI). The signalP server (htpp://www.cbs.dtu.dk/signalp/cbssignalp.html) was used to identify the predicted cleavage site of the signal peptide for CLF-1. DNA and amino acid sequence alignments as well as prediction of hydrophobic regions were analyzed with the Wisconsin package version 8.1 (Genetics Computer Group, Madison, WI).
Source of cells and culture conditions
The cell lines HEK-293 and CHO were obtained from the American Type Culture Collection (Manassas, VA). Palmar fibromatosis lesion and mammary gland fibroblasts were gifts from Prof. G. Gabbiani (University Medical Center, Geneva, Switzerland). Tonsillar fibroblasts were derived from whole tonsils obtained from surgery following a previously described protocol (27). All the above cell types were maintained in DMEM and Ham’s F-12 nutrient solution supplemented with 10% FCS. Fibroblastic cells and cell lines were stimulated with the appropriate cytokines at a final concentration of 10 ng/ml/cytokine.
Detection of the CLF-1 gene by Southern blot analysis
Genomic DNA was isolated from cell lines or tissues with the DNAzol reagent (Life Technologies, Grand Island, NY) following the manufacturer’s guidelines. The source of the genomic DNA was as follows: HEK-293 human fibroblastic cells, African green monkey COS-7 cells (American Type Culture Collection), C57BL/6 mouse tails, rat liver, rabbit liver, cow liver, and chicken embryo liver. Aliquots of genomic DNA (5 µg) were digested with either SacI or BamHI and subjected to Southern blot analysis using a 32P-labeled 1.2-kb fragment of hCLF-1 cDNA as a probe. Hybridization was performed in 5x SSC, 5x Denhardt’s solution, and 5% SDS at 55°C. Filters were washed with 0.1x SSC and 1% SDS at 55°C and exposed at -70°C.
Detection of hCLF-1 mRNA by Northern blot analysis
The same 32P-labeled 310-bp hCLF-1 cDNA fragment that was used to screen the human placental cDNA library (see above) was used to identify hCLF-1 mRNA transcripts in the Human, Human II, Human Immune System, Human Endocrine, and Human Fetal Multiple Tissue Northern Blots (Clontech). Hybridization was performed in ExpressHyb solution (Clontech). Hybridization and washing were performed according to the manufacturer’s guidelines.
Detection of hCLF-1 mRNA by RT-PCR
Total RNA was purified from the appropriate source using TRIzol reagent solution (Life Technologies) following the manufacturer’s guidelines. Five micrograms of total RNA was reverse transcribed using the first strand cDNA synthesis kit (Pharmacia LKB Biotechnology, Uppsala, Sweden) following the manufacturer’s guidelines, and 1/10th of the cDNA was amplified by 32 cycles of PCR using the primers 5'-GGATCAGGAGCCCACACAGC-3' and 5'-AGCGGCAGGTCAAGTCCTTC-3' for hCLF-1 cDNA and 5'-GGCGACGAGGCCCAGAGCAAG-3' and 5'-CGATTTCCCGCTCGGCCGTG-3' for ß-actin cDNA. PCR products were analyzed by agarose gel electrophoresis.
Production of recombinant soluble hCLF-1
To generate recombinant soluble protein, hCLF-1 cDNA in pBluescript II SK- was digested with EcoRI and StuI and cloned into pFASTBAC-1 (Life Technologies) digested with the same enzymes. The oligonucleotides 5'-CGCGTGCCTCGAACCCTACACCGCCTGCGACCATCACCATCACCATCACTGA-3' and 5'-TCAGTGATGGTGATGGTGATGGTCGCAGGCGGTGTAGGGTTCGAGGCA-3' were annealed and cloned into pFASTBAC-1 containing the hCLF-1 cDNA digested with MluI and StuI. The annealed oligonucleotides encoded six histidines and the CLEPYTACD tag, an epitope recognized by mAb 179 (Affymax, Palo Alto, CA), at the 3' end of the hCLF-1 cDNA.
Recombinant baculovirus was generated using the BAC-TO-BAC kit (Life Technologies) and was used to infect Sf9 cells (American Type Culture Collection) expanded in SF900II medium (Life Technologies). Recombinant protein was purified from the Sf9 culture medium using a column packed with 50 ml of Ni-NTA (Qiagen, Basel, Switzerland) following the manufacturer’s guidelines. The recovered fraction contained approximately 30% monomeric hCLF-1 as determined by SDS-PAGE under reducing conditions.
Expression of cell membrane-bound hCLF-1 in Sf9 cells
To generate membrane-bound hCLF-1, pBluescript II
SK- containing hCLF-1 cDNA was digested with
SacII. The human IL-13R1 (hIL-13R1) (28) transmembrane
and cytoplasmic domains were amplified by PCR from
pBluescript II SK- containing the hIL-13R1 cDNA using
the oligonucleotides 5'-TCCCCGCGGTACATAACCATGTTACTCATTGTT-3' and
5'-TCCCCGCGGGAA TTCCATCACTGAGAGGCTTTCTT-3'. The PCR product was
digested by SacII and ligated into the
SacII-digested pBluescript II SK- containing
hCLF-1 cDNA. DNA encoding the polyoma virus middle T Ag epitope EYMPME
(EE tag) (29) was inserted at the 5' end of the hCLF-1 cDNA by
digesting the plasmid with XhoI and BamHI and
cloning into these restriction enzyme sites the two annealed
oligonucleotides
(5'-TCGAACTAGTGAATACATGCCAATGGAAGCCCACACAGCTGTGATCAGTCCCCAG-3' and
5'-GATCCTGGGGACTGATCACAGCTGTGTGGGCTTCCATTGGCATGTATTCACTAGT-3').
The cDNA encoding the tagged fusion protein was excised from
pBluescript II SK- by digestion with SpeI
(whose site was subsequently blunt ended using the Klenow fragment of
DNA polymerase I) and EcoRI, and ligated into pFASTBAC-1
(containing the mellitin signal peptide) digested with
Bsp120I (whose site was subsequently blunt ended using the
Klenow fragment of DNA polymerase I) and EcoRI.
Recombinant baculovirus was generated using the BAC-TO-BAC kit and used to infect Sf9 cells. Expression of the fusion protein on the surface of the Sf9 cell line was confirmed at 24 and 48 h by flow cytometry using a mAb recognizing the EE tag. Briefly, cells were washed with FACS buffer (1% BSA and 0.01% sodium azide in PBS) and successively incubated for 30 min with the anti-EE mAb at 10 µg/ml in FACS buffer and FITC-labeled sheep anti-mouse F(ab')2 fragments (Silenius Laboratories, Hawthorn, Australia) diluted 1/100 in FACS buffer. Fluorescence was measured using a FACSCalibur (Becton Dickinson, Erembodeggen, Belgium).
Expression of cell membrane-bound hCLF-1 in CHO cells
To generate membrane-bound hCLF-1 expressed in mammalian cells,
pBluescript II SK- containing the hCLF-1/hIL-13R1 cDNA
without the EE tag was digested with BamHI and
EcoRI and inserted into the expression vector pCDNA3
(Invitrogen) at the same restriction enzyme sites. To insert the native
signal peptide from hCLF-1 at the 5' end of the fusion construct,
pBluescript II SK- containing the full-length hCLF-1 was
digested with KpnI and BamHI, and the excised
fragment (encoding the signal peptide) was inserted into pCDNA3
containing the hCLF-1/hIL13R1 cDNA digested with the same enzymes.
The expression vector was linearized and introduced into CHO cells by
electroporation using previously described conditions (28).
Transfected cells were selected using 500 µg/ml geneticin (Life
Technologies). Control cells were transfected with pCDNA3 containing
the full-length hIL-13R1 cDNA. Expression of hCLF-1 on the surface
of the CHO cells was detected by flow cytometry with a mAb recognizing
hCLF-1 (see below) using the conditions described above. Positive cells
were selected and sorted by flow cytometry and replaced in culture.
After three rounds of sorting, a population of CHO cells expressing
high levels of membrane-bound hCLF-1 was obtained.
Generation of mouse anti-hCLF-1 mAbs
A BALB/c mouse was immunized on days 0, 7, and 28 s.c. in the limbs and behind the neck with 100 µg of purified recombinant hCLF-1 in MPL+TDM emulsion (RIBI, Inotech, Dottikon, Switzerland) per injection. Three days after the final injection, the draining lymph nodes were obtained, and the tissue was digested using a DNase and collagenase mixture as reported previously (30). The resulting cell suspension was resuspended at 106 cells/ml and fused with Sp2 myeloma cells using a standard protocol (31). The hybridomas were selected in hypoxanthine-aminopterin-thymidine medium, and 7 to 10 days after fusion, the supernatants were harvested for screening.
Screening hybridoma supernatants for anti-hCLF mAbs
Ninety-six-well plates (Falcon 3912, Becton Dickinson Labware
Europe, Meylan, France) were coated at 4°C with 1 µg/ml recombinant
hCLF-1 in carbonate buffer, pH 9.6. Plates were washed, blocked with
1% BSA in PBS, incubated for 2 h with 200 µl of hybridoma
supernatant, and washed again. Bound Ab was revealed with horseradish
peroxidase-coupled goat anti-mouse IgG (Southern Biotechnology
Associates, Birmingham, AL). Specificity was checked using an ELISA
with recombinant soluble
hIL-13R16 that had the
same recognition tags as recombinant hCLF-1 at 1 µg/ml. Specific
positive supernatants were further screened by flow cytometry on Sf9
cells either mock infected or infected with baculovirus encoding the
membrane-bound hCLF-1/hIL13R1 fusion protein or on transfected CHO
cells expressing hIL-13R1 or the hCLF-1/hIL-13R1 fusion protein.
Purification of anti-hCLF-1 mAbs
Abs were purified by chromatography on protein A-Sepharose Fast Flow in PBS and eluted in 0.1 M citrate, pH 4.5. Eluates were then subjected to gel filtration on Superdex-200 (Pharmacia) equilibrated in PBS.
Detection of hCLF-1 by Western blot analysis
The supernatants or cell lysates containing the recombinant protein were diluted 1/1 with Tris-glycine-SDS sample buffer (Novex, San Diego, CA) and heated to 95°C for 5 min. To reduce proteins, ß-ME was added to the samples to a final concentration of 375 mM. The proteins were resolved on an 8% polyacrylamide gel (Novex) and electrotransferred onto a nitrocellulose membrane (32). The membrane was blocked in PBS containing 5% dried milk and 0.15% Tween-20 for 1 h at room temperature and incubated for 1 h at room temperature with PBS containing 2.5% dried milk and 5 µg/ml of the appropriate mAb. Bound mAb was detected with horseradish peroxidase-labeled sheep anti-mouse Ab and ECL (Amersham Life Sciences, Aylesbury, U.K.) following the manufacturer’s guidelines.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The GenBank database with ESTs was searched using TBLASTN with a
20-amino acid sequence surrounding the W-S-x-W-S motif of murine IL-13R
(26). ESTs showing significant homology were then translated, and the
open reading frames were used to search the Swissprot database using
BLASTP for homologous proteins. The amino acid sequence from the murine
EST W66776 shared a high level of homology with the prolactin receptor
(33) and the receptors for the IL-6-type cytokines (10). Using the
sequence of W66776 to search the GenBank database allowed the
identification of overlapping homologous sequences (of both murine and
human origin), which, in turn, were run against the GenBank database to
identify more overlapping ESTs (Table I
).
This allowed the assembly of overlapping sequences encoding the human
and mouse cDNAs. The cDNA clone 479043, which gave rise to the mouse
EST found furthest 5' in the sequence assembly was obtained from the
IMAGE consortium. After sequencing, it was found to contain an insert
of 1 kb, including a 3' poly(A) tail. The rapid amplification of 5'
cDNA ends on murine lung cDNA allowed the cloning of a further 308 bp
upstream.
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The murine cDNA encoded a protein of 383 amino acids. The cDNA was
incomplete at the 5' end as the first amino acid of the translated
sequence aligned to amino acid 39 of the human sequence, and no
starting methionine or putative signal peptide could be identified.
Both mouse and human CLF-1 contained 11 cysteine residues and 6
potential N-linked glycosylation sites. Sequence analysis of
the human and murine cDNAs showed 85% nucleic acid identity and 96%
amino acid identity (Fig. 1A).
Human and murine CLF-1 showed close homology to the prolactin receptor
and receptors of the IL-6-type cytokines (10) (Table II). The N-terminal region of both
sequences appeared to represent an Ig-like domain, most closely
resembling the C2-set sequence (34). This domain was followed by two
fibronectin type III modules of approximately 100 amino acid residues
each. Alignment of the human and mouse amino acid sequences to members
of the IL-6-type cytokine receptor family showed regions of conserved
homology within these two functionally important modules known as the
cytokine receptor-like domain, and both sequences contained the highly
conserved four cysteine residues and the W-S-x-W-S motif characteristic
of this domain (5) (Fig. 1B). We were unable to identify any
transmembrane domains within the amino acid sequence of the mature
proteins or hydrophobic region at the C-terminus of the sequence,
characteristic of glycosyl phosphatidylinositol-anchored proteins
such as the CNTFR -chain (CNTFR) (35). This suggested that the
cloned human and mouse cDNAs encode soluble proteins.
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Using the hCLF-1 cDNA sequence to search the GenBank database we
identified the hCLF-1 gene sequence, under the accession number
AC003112. The gene was encoded by nine exons contained within a 14-kb
region of the chromosome 19-specific cosmid R30292 mapping to 19p12
(confirming the location predicted by the EST with accession no.
H14009). The erythropoietin receptor (36), sharing homology with CLF-1,
and the soluble protein EBI3 (37) are the only known members of the
cytokine type I receptor family shown to be localized on this arm of
chromosome 19. The intron/exon organization of the gene for hCLF-1 is
in agreement with the predicted structure of the domains within the
hCLF-1 protein, following the rule proposed for the cytokine
receptor-like domain (Fig. 2) (38). The
first exon encodes the signal peptide, exon 2 encodes the Ig-like
domain, exons 3 and 4 encode the N-terminal cytokine receptor-like
domain, and exons 5 and 6 encode the C-terminal cytokine receptor-like
domain. Exons 7 and 8 showed no homology with any other family members,
while exon 9 had the stop codon close to its 5' end and the
polyadenylation signal close to its 3' end. We were unable to identify
anything resembling an exon encoding a putative transmembrane domain
within the 17.4 kb of DNA sequence contained within the contig
downstream of the exon containing the W-S-x-W-S motif (exon 6).
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The very high amino acid identity between the human and mouse
protein sequences for CLF-1 suggested that the gene had been highly
conserved during recent evolution. As the cDNAs encoding human and
mouse CLF-1 shared 85% homology, we examined the conservation of the
gene in a number of animal species by Southern blot analysis of genomic
DNA using an hCLF-1 cDNA probe (Fig. 3).
As expected, human and monkey genomic DNA gave the strongest signals,
but signals could be detected in all six mammalian species studied,
with faint bands also seen with chicken genomic DNA, indicating that
the gene is highly conserved within these species.
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hCLF-1 mRNA expression was studied in human tissues by Northern
blot analysis. The mRNA transcript migrated as a 1.7-kb species, a size
close to that predicted from the clone obtained by library screening.
Expression of the 1.7-kb transcript could be detected in several
tissues (Fig. 4). The strongest
expression of the hCLF-1 mRNA was detected in the spleen, thymus, lymph
node, appendix, bone marrow, stomach, placenta, heart, thyroid, and
ovary. Interestingly, a strong level of expression was also detected in
fetal lung. We studied the level of mRNA expression by Northern blot
analysis in several cell lines, including the fibroblastic cell line
HEK 293, the monocyte cell line THP-1, JY lymphoblastoid cells, RPMI
8226 myeloma cells, the mast cell line HMC-1, HBE-140 bronchial
epithelial cells, and HUVEC. A detectable level of expression could be
seen in JY cells, HMC-1 cells, and PMA-stimulated THP-1 cells. The
strongest expression, however, was detected in HEK-293 cells (data not
shown).
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A), an up-regulation of
hCLF-1 mRNA was observed in all three fibroblast primary cell cultures
under stimulation with IFN-. TNF- up-regulated hCLF-1 mRNA in
palmar fibromatosis lesion and mammary gland fibroblasts, while IL-6
up-regulated hCLF-1 mRNA in palmar fibromatosis lesion and tonsillar
fibroblasts. Interestingly, IL-1ß had a down-regulatory effect on
hCLF-1 mRNA production in two of the three fibroblast cultures (Fig. 5, B–D).
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A BALB/c mouse was immunized with recombinant soluble hCLF-1
generated with the baculovirus expression system. Supernatants from the
resulting hybridomas were tested by ELISA using the same recombinant
protein. An ELISA using a recombinant soluble form of hIL-13R1 with
the same recognition tags as the recombinant hCLF-1 was performed on
the positive hybridomas to eliminate Abs raised against the tags or
conserved cytokine receptor epitopes. Specific positive mAbs were then
tested by flow cytometry using Sf9 cells infected with baculovirus
encoding the hCLF-1/hIL-13R1 membrane-bound fusion protein or CHO
cells stably transfected with an expression vector encoding the same
fusion construct. A strong signal was seen with one hybridoma,
92A10, with no signal detected on mock-transfected Sf9
cells or hIL-13R1-transfected CHO cells (Fig. 6). The mAb expressed by this hybridoma
was determined to be an IgG2a.
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The recombinant forms of hCLF-1 expressed in Sf9 and CHO cells
were examined by Western blot analysis under nonreducing and reducing
conditions using either the appropriate anti-tag mAb or the
anti-hCLF-1 mAb. The mAb 179 revealed that in the supernatant of
Sf9 cells infected with baculovirus encoding recombinant soluble
hCLF-1, the protein formed covalently linked tetramers. This was
demonstrated by the presence of a high molecular mass band (
200 kDa)
detected under nonreducing conditions. Under reducing conditions, only
the band corresponding to monomeric protein could be detected
(48–49 kDa; Fig. 7A). A
similar pattern was detected using the anti-EE mAb on the cell
lysate of baculovirus-infected Sf9 cells expressing the
hCLF-1/hIL-13R1 fusion protein. Western blotting revealed a
predominant band corresponding to covalently linked homodimeric protein
(120 kDa) when performed under nonreducing conditions. A larger band
with a molecular mass of >200kDa was also detected, probably
corresponding to covalently linked tetrameric protein. Under reducing
conditions, the only band detected was that which corresponded to the
monomeric form of the protein (60 kDa; Fig. 7B). The
hCLF-1/hIL-13R1 fusion protein was detected in the cell lysate of
stably transfected CHO cells using the anti-hCLF-1 mAb. Again,
under nonreducing conditions the protein formed covalently linked
homodimers of 130 to 150 kDa (Fig. 7C).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Human and murine CLF-1 were seen to share 96% amino acid identity
(Fig. 1A) and also show highest homology with the prolactin
receptor (33) and gp130 (12). It is interesting to note that amino acid
identity between human and mouse gp130 is 77%, and that between the
human and mouse prolactin receptors is 69%. Southern blot analysis,
using hCLF-1 cDNA as a probe, indicated that the high conservation of
the CLF-1 gene sequence between human and mouse can be extended to
monkey, rat, rabbit, cow, and chicken (Fig. 3). The hybridization and
washing conditions were relatively stringent, thus reducing the risk of
nonspecific signals. This extremely high level of conservation during
recent evolution suggests a functionally important role for CLF-1.
Information concerning the homology between the N-terminal region of
human and mouse CLF-1 and the extracellular region of members of the
cytokine type I receptor family is provided in Figure 1B and
Table II. The four conserved cysteines and the W-S-x-W-S motif are
shown above the alignment in Figure 1B. These sequence
motifs characterize members of the type I cytokine receptor family (5)
and are located within two fibronectin type III modules (7) that have
been shown to play an important role in receptor function (8, 9). This
homology suggests that CLF-1 constitutes a new member of the cytokine
type I receptor family. The CLF-1 gene structure is also typical of
those found within this family of receptors (Fig. 2) (38).
Although CLF-1 appears to exist uniquely as a soluble protein, there
are numerous examples of soluble forms of receptor subunits in the
cytokine type I receptor family. These soluble subunits can exhibit
either antagonistic effects in terms of ligand signaling, such as those
shown by soluble gp130 (39, 40) and the soluble IL-5R chain
(IL-5R) (41), or agonistic effects, such as those shown by the
IL-6R chain (IL-6R) (16, 17), the IL-11R chain (IL-11R)
(18), and CNTFR (19). Receptor chains within the cytokine type I
receptor family generally function as receptor signaling or ligand
binding subunits (11). If we are to assume that CLF-1 exists solely in
soluble form and is indeed a soluble receptor chain, it is likely that
it serves as a ligand binding subunit. Further analysis must be
undertaken to determine whether any of the known ligands is able to
bind CLF-1.
Human CLF-1 mRNA is predominantly expressed in adult spleen, thymus,
lymph node, appendix, bone marrow, stomach, placenta, heart, thyroid,
and ovary (Fig. 4). We can also predict that the transcript is well
expressed in pregnant female uterus when looking at the source of
origin of hCLF-1 ESTs (Table I). Expression of hCLF-1 mRNA was also
studied by Northern blot analysis in a number of different primary and
transformed cells. Strongest expression was observed in the
fibroblastic cell line HEK 293 with detectable levels in the B cell
line JY, the immature mast cell line HMC-1, and the monocyte cell line
THP-1 following stimulation (data not shown). The expression seen in
HEK 293 cells prompted us to study the modulation of hCLF-1 mRNA levels
in HEK 293 cells and fibroblast primary cell cultures in response to
the proinflammatory cytokines IL-1ß, TNF-, IL-6, and IFN-.
Although these cytokines had no real effect on the constitutive level
of hCLF-1 mRNA expression in HEK 293 cells, a significant up-regulation
of mRNA was observed in the primary fibroblast cultures in response to
IFN- with an up-regulation in two of the three cultures in response
to TNF- and IL-6, as detected by RT-PCR (Fig. 5, A–D).
The tissue distribution of hCLF-1 mRNA and its up-regulation by
proinflammatory cytokines in fibroblast cultures suggest that hCLF-1
may be involved in regulation of the immune system during an
inflammatory response.
A strong expression of hCLF-1 mRNA was also observed in fetal lung
(Fig. 4). In addition, mCLF-1 mRNA transcripts were detected by
Northern blot analysis in total mouse embryos on days 11, 15, and 17
days postcoitum, with no expression seen on day 9 postcoitum (data not
shown). Furthermore, many ESTs encoding human and mouse CLF-1 were
derived from fetal tissues (Table I). This points to an additional role
for CLF-1 in mediating regulatory signals during fetal development.
Western blot analysis of the recombinant forms of hCLF-1
baculovirus-infected Sf9 cell culture supernatants, membrane-bound
hCLF-1 fusion protein from baculovirus-infected Sf9 cell lysates and
membrane-bound hCLF-1 fusion protein from stably transfected CHO cell
lysates under nonreducing and reducing conditions, provided two
significant observations (Fig. 7, A–C). Firstly, the
recombinant protein had a tendency to exist in covalently linked
homomeric forms when comparing the difference in molecular mass under
nonreducing or reducing conditions. Secondly, it appeared that the
anti-hCLF-1 mAb recognized the nonreduced form of the protein,
while reduced (i.e., monomeric) forms of the protein were poorly
recognized. These findings suggested that the mAb bound to a structural
epitope, formed when two or more hCLF-1 chains are covalently linked
together, as opposed to a sequence epitope on the protein. Furthermore,
HEK 293 cells (which constitutively expressed hCLF-1 mRNA) were used in
an attempt to detect hCLF-1 protein in its native form using the mAb
raised against the recombinant protein. No evidence was obtained of
hCLF-1 being expressed on the cell surface by flow cytometry or in the
cell supernatant by Western blot or immunoprecipitation, suggesting one
of three possibilities. Firstly, a tight translational control could
result in a level of protein production below the threshold of
detection of the Ab. Secondly, hCLF-1 could be secreted from the cell
in monomeric form, rendering itself undetectable to the mAb. Thirdly,
hCLF-1 could form a covalently linked heterodimeric structure with a
different subunit, again rendering itself invisible to the mAb.
The cytokine IL-12 is composed of two disulfide-linked
N-glycosylated polypeptides of approximately 40 kDa (p40)
and 35 kDa (p35). Certain regions of the p35 amino acid sequence show
homology to IL-6 and granulocyte CSF, suggesting that p35 is derived
from a cytokine-like molecule. The p40 amino acid sequence, however,
shows homology with the extracellular domain of members of the cytokine
type I receptor family, most notably with IL-6R and CNTFR.
Interestingly, recombinant p40 can form a covalently linked homodimer,
a form that in the mouse acts as a physiologic antagonist of IL-12,
probably competing for IL-12 binding to its receptor (24, 42, 43).
From these observations, it is tempting to compare CLF-1 with IL-12
p40. Firstly, both proteins share homology with the extracellular
regions of IL-6R and CNTFR. Secondly, when recombinant protein is
expressed in cells transfected with the appropriate cDNA, both CLF-1
and IL-12 p40 can form covalently linked homodimers. If, indeed, as
with the IL-12 p40 subunit, CLF-1 exists in its native functional form
as a covalently linked heterodimer, this could well account for our
failure to detect the protein using the anti-hCLF-1 mAb in cells
expressing hCLF-1 mRNA as discussed earlier.
The generation of Abs recognizing the native form of CLF-1 could allow the purification and thus structural characterization of the native protein, leading to the identification of covalently linked heterologous subunits. The identification of a cell membrane-bound counterstructure for CLF-1 is ongoing and will also further the characterization of this novel protein.
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Acknowledgments |
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Footnotes |
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2 Current address: Centre d’Immunologie Pierre Fabre, 74164 Saint-Julien-en-Genevois, France.
3 Current address: Serono Pharmaceutical Research Institute, Ares Serono Research and Development, 1228 Plan-les-Ouates, Switzerland.
4 Address correspondence and reprint requests to Dr. M. H. Kosco-Vilbois, Serono Pharmaceutical Research Institute, 14 Chemin des Aulx, 1228 Plan-les-Ouates, Switzerland.
5 Abbreviations used in this paper: CNTF, ciliary neurotrophic factor; LIF, leukemia inhibitory factor; CNTFR, ciliary neurotrophic factor receptor; CLF-1, cytokine-like factor-1; EST, expressed sequence tag; hCLF-1, human cytokine-like factor-1; mCLF-1, murine cytokine-like factor-1; hIL-13R1, human IL-13 receptor 1-chain.
6 P. Graber et al. The distribution of IL-13R1 expression by B cells, T cells and monocytes and its regulation by IL-13 and IL-4. Submitted for publication.
Received for publication March 9, 1998. Accepted for publication March 30, 1998.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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component as a soluble mediator of CNTF responses. Science 259:1736. mRNA expressed in human B, T, and endothelial cells encoding an alternate type-II interleukin-4/interleukin-13 receptor. Eur. J. Immunol. 27:971.[Medline]
subunit of the human granulocyte-macrophage colony stimulating factor receptor: implications for the evolution of the cytokine receptor superfamily. J. Biol. Chem. 269:10905. chain and a ß chain shared with the receptor for GM-CSF. Cell 66:1175.[Medline]
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