Cloning and expression of the first nonmammalian
interleukin-11 gene in rainbow trout Oncorhynchus mykiss
Tiehui Wang,
1
Jason W. Holland,
1
Niels Bols
2
and Christopher J. Secombes
1
1 Scottish Fish Immunology Research Centre, University of Aberdeen, UK
2 Department of Biology, University of Waterloo, Ontario, Canada
Interleukin (IL)-11 is a 19 kDa, highly conserved, non-
glycosylated protein that was originally identified and
cloned from a primate bone marrow-derived stromal
cell line (PU-34) as a lymphopoietic and hemotopoietic
cytokine [1]. It is synthesized as a 199 amino acid pre-
cursor and secreted as a 178 amino acid mature pep-
tide after cleavage of the 21 amino acid signal peptide
[2]. It is unusually basic for a cytokine, and has a high
content of proline, leucine and positively charged
amino acids. IL-11 is thought to exist as a thermally
stable, four-helix bundle structure, although it contains
no cysteine residues [3]. It is a member of the gp130
family of cytokines that includes IL-6, leukaemia
inhibitory factor, oncostatin M, cardiotropin 1, ciliary
Keywords
Aeromonas salmonicida; cloning;
expression; interleukin-11; rainbow trout
Correspondence
C. J. Secombes, Scottish Fish Immunology

glycosylation site that are not present in mammals. Phylogenetic analysis
clearly grouped trout IL-11 with IL-11 molecules from other species and
separated from other members of the IL-6 family. The IL-11 gene is highly
expressed in intestine and gills in healthy fish and its expression can also be
detected in spleen, head kidney, brain, skin and muscle. Bacterial infection
of rainbow trout markedly up-regulates IL-11 expression in liver, head
kidney and spleen. IL-11 expression is also up-regulated in RTS-11 cells
(a trout macrophage cell line), which constitutively expressed the lowest
level of IL-11 of the four trout cell lines examined, after stimulation with
bacteria, lipopolysaccharide, poly(I:C) and recombinant trout IL-1b. Only
a single transcript of 3.2 kb could be detected in lipopolysaccharide or
recombinant IL-1b-stimulated RNA samples by northern blotting. The
expression results, showing that IL-11 is widely distributed and modulated
by infection and other cytokines, suggest that fish IL-11 is an active player
in the cytokine network and the host immune response to infection.
Abbreviations
IL, interleukin; LPS, lipopolysaccharide; MOI, multiplicity of infection; ORF, open reading frame; pI, isoelectric point; TGF, transforming
growth factor; TNF, tumour necrosis factor; UTR, untranslated region.
1136 FEBS Journal 272 (2005) 1136–1147 ª 2005 FEBS
neurotrophic growth factor and a viral homologue of
IL-6 encoded by the Kaposi’s sarcoma-associated her-
pesvirus. All of these cytokines elicit either hetero- or
homodimerization of gp130, which activates intracellu-
lar signal transduction pathways [4]. IL-11 has three
receptor-binding sites, with a signal being initiated by
binding to the specific IL-11R via site I and gp130 via
sites II and III, through the formation of a hexameric
receptor complex consisting of two molecules each of
IL-11, IL-11R, and gp130 [5].
IL-11 is produced by many cell types throughout

stimulates osteoclast formation and bone resorption
in vitro [15]. Indeed, transgenic overexpression of
IL-11 stimulates osteoblastogenesis and bone forma-
tion [16]. Thus, IL-11, together with other members
of the gp130 family, are essential for bone metabo-
lism [17]. Lastly, IL-11 signalling is an absolute
requirement for normal development of placentation
and survival to birth [18]. Although IL-11R– ⁄ – mice
display normal hematopoiesis [19], female IL-11R– ⁄ –
mice are infertile because of defective decidualization
[20] and mutant mice with low IL-11R activity have
low fertility [21].
Owing to its key activities on thrombocytopoiesis
and the development of placentation, IL-11 is currently
believed to be restricted to mammals and to date no
nonmammalian IL-11 molecules have been described.
In an effort to identify immune genes involved in host
defence against bacterial infection in rainbow trout
(Oncorhynchus mykiss), the gene-expression profile of
bacterial-challenged rainbow trout was surveyed by
means of suppression subtraction hybridization and
sequence analysis [22]. This resulted in identification of
a number of immune genes, including a SSH clone
with homology to mammalian IL-11. As the expression
of this clone was highly induced in tissues of bacterial
infected fish, a full-length cDNA clone as well as a
genomic DNA clone were isolated and sequenced, and
the expression and modulation of this molecule was
studied.
Results

trout IL-11 gene has a five exon ⁄ four intron organiza-
tion and all introns are phase 0 except intron I, which
T. Wang et al. Rainbow trout interleukin-11
FEBS Journal 272 (2005) 1136–1147 ª 2005 FEBS 1137
Rainbow trout interleukin-11 T. Wang et al.
1138 FEBS Journal 272 (2005) 1136–1147 ª 2005 FEBS
is phase 1 (Fig. 1). This exon ⁄ intron organization
resembles that of mammalian IL-11. The sequences
between the trout cDNA and genomic DNA in the
coding region are identical, although there were differ-
ences in both the 5¢- and 3¢-UTR. The major differ-
ences were a 26 bp insertion in the 5¢-UTR of the
cDNA and an insertion of 12 repeats with a consensus
of CCAATGATGATCCAAGAAATCCACACTACAG
(31 bp) in the 3¢-UTR of the cDNA sequence (Fig. 1).
A TATA box was identified 28 bp upstream from the
cDNA sequence.
The trout IL-11 translation showed only 32 ⁄ 40%
sequence identity ⁄ similarity to primate counterparts,
and even lower homology (29 ⁄ 39% identity ⁄ similarity)
to rodent IL-11, whereas mammalian IL-11 molecules
share high sequence identities, with 94.5% identity
between primates, 97% identity between rodents,
and 84% identity between primates and rodents
(Table 1). When preparing this article, an IL-11
homologue in the fugu (Tetraodon nigroviridis) data-
base was discovered. The trout translation showed
similar homology to the fugu IL-11 molecule as to
mammalian molecules. However, the trout molecule
shares a higher nucleotide identity of 46.7% in the

Leucine
a
(%) 15.4 (28) 13.5 (24) 23.0 (41) 23.6 (42) 21.9 (39) 21.3 (38)
Positive charged
a
(%) 9.9 (18) 12.4 (22) 11.8 (21) 11.8 (21) 11.8 (21) 11.8 (21)
Isoelectric point 7.91 9.83 11.16 10.71 11.16 11.16
a
Percentage of amino acid in the mature peptide. Number in brackets is actual number of residues present.
Fig. 1. Nucleotide and deduced amino acid sequences of the trout IL-11 gene. The genomic DNA (upper line, accession number 867256),
cDNA (middle line, accession number AJ535687) and amino acid (lower line) sequence are numbered on the left according to the submitted
sequences. The 5¢-and3¢-flanking and intron sequences are in lowercase. Identical nucleotides in the cDNA to genomic DNA sequence is
relaced by ‘|’ and insertions indicated by ‘-’. The two insertions in the 5¢- and 3¢-UTR are underlined and the 13 repeats with a consensus of
CCAATGATGATCCAAGAAATCCACACTACAG (31 bp) in the 3¢-UTR are numbered and distinguished from each other by alternate highlighting
in bold and italics. The ATTTA motifs in the 5¢-and3¢-UTRs, the start and stop codons, the signal peptide predicted by
SIGNALP 3.0 [23] and
the potential N-glycosylation site (NQT) are in bold and underlined. The four potential poly(A) signals (AATAAA) in the 3¢-UTR and the TATA
box in the 5¢-flanking region are boxed.
T. Wang et al. Rainbow trout interleukin-11
FEBS Journal 272 (2005) 1136–1147 ª 2005 FEBS 1139
with 21 amino acids in mammals. There are cysteine
residues (one in trout and four in fugu) and potential
N-glycosylation sites (one in trout and two in fugu)
present in mature peptides of the fish IL-11s that
are not present in mammals (Table 2). The two fish
IL-11s have a lower leucine content; 15.4% in fugu
and 13.5% in trout, compared with 21.3–23.6% in
mammals. Trout and mammalian IL-11s have a high
proline content, positively charged amino acid content
and high isoelectric points, in contrast to fugu IL-11

as shown in Fig. 4 in liver, head kidney and spleen tis-
sues. No IL-11 expression was detectable using 28
cycles in samples prepared from control fish or phos-
phate-buffered saline (PBS)-injected fish, however, a
PCR product was just detectable in samples from fish
injected with bacteria 6 h after infection, and a strong
product was present in samples prepared at 24 and
48 h after infection (Fig. 4).
Expression and modulation of trout IL-11
in cell lines
The constitutive expression of IL-11 was examined
in four trout cell lines from samples prepared from
Fig. 2. Multiple alignment of the predicted
rainbow trout IL-11 translation with known
IL-11 molecules. Identical (*) and similar resi-
dues (: or.) identified using
CLUSTAL W are
indicated. The signal peptides are in bold.
The potential N-glycosylation sites and cys-
teine residues present in the two fish mole-
cules, and the three conserved residues
(K42, M59 and K99) in the mammalian
mature IL-11 protein, are in bold and under-
lined. The conserved residues L67 and R169,
critical to binding IL-11R, are boxed. A, B, C
and D indicate the four alpha helices [5].
Accession numbers are: human, P20809;
monkey, P20808; mouse, P47873; rat,
Q99MF5; puffer fish, Q6UAM0 and trout,
AJ535687.

blotting. A single transcript of  3.2 kb was detectable
that was highly expressed in LPS- and recombinant
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Fig. 3. A phylogenetic tree of trout IL-11 and other members of the IL-6 family cytokines including IL-6, IL-11, LIF (leukaemia inhibitory fac-
tor), OSM (Oncostatin M), CT-1 (cardiotrophin-1) and CNTF (ciliary neurotrophic factor). A carp molecule (M16) that shares sequence similar-

24 h
48 h
6 h
24 h
48 h
DNA marker
Control
Neg. control
Fig. 5. Expression of trout IL-11 in vivo in
bacterially challenged rainbow trout. Trout
were injected with PBS or bacteria (Aero-
monas salmonicida, MT423) and three fish
were sampled at 6, 24 and 48 h postinjec-
tion, as described in the Experimental proce-
dures. Three healthy fish were killed for the
control samples. Total RNA from three fish
at each time point was pooled and used for
RT-PCR analysis. The cycle numbers were
21 and 28 for b-actin and IL-11, respectively.
IL-11
123 123123123 123123 123123
Muscle
Head kidney
Spleen
Liver
Gills
Skin
Intestine
Brain
100 bp

requirement for IL-11 in normal development of plac-
entation in mammals is a biological activity that is
apparently not needed in fish. However, other func-
tions, such as anti-inflammatory effects and other
hematopoietic effects, might be conserved and await
functional studies with the recombinant protein.
IL-11 expression is regulated mainly at the post-tran-
scription level, by stabilizing the transcribed mRNA
that possesses multiple copies of a destabilizing signal
(ATTTA) in the 3¢-UTR [28]. Multiple ATTTA motifs
are also present in the 3¢-UTR of trout and pufferfish
IL-11 sequences, as well in the 5¢-UTR of the trout
transcript. Human and monkey IL-11 is produced as
two mRNA transcripts of 1.5 and 2.5 kb which differ
at their 3¢-polyadenylation sites yet encode the same
Marker
Control
3 h
7 h
24 h
3 h
7 h
24 h
3 h
7 h
24 h
Neg. Control
Marker
LPS MT423 Poly I:C
β-actin

PCR were 21 and 30 for b-actin and IL-11,
respectively.
β-actin
IL-11
3.2 kb
1.8 kb
Control
LPS
LPS
β-actin
IL-11
3.2 kb
1.8 kb
Control
rIL-1
β
rIL
-1
β
A
B
Fig. 7. Northern blot analysis of trout IL-11 expression in RTS-11
cells stimulated for 4 h with LPS (25 lgÆmL
)1
) (A) and recombinant
trout IL-1b (25 ngÆmL
)1
) (B). Twenty micrograms of RNA was sep-
arated, transferred to a Hybond nylon membrane, and hybridized
with

an anti-inflammatory cytokine. Although in healthy
fish IL-11 is expressed at very low levels in liver, head
kidney and spleen, as evidenced from the high cycle
number of 32 needed for its detection by RT–PCR, its
expression is highly induced in these tissues after bac-
terial infection. This induction of expression has also
been seen after infection in mammals where, for exam-
ple, human cord blood-derived macrophages and dend-
ritic cells express a higher level of IL-11 upon virus
infection [6]. IL-11 mRNA up-regulation has also been
seen in mice after infection with Pseudomonas aerugi-
nosa [7]. As IL-11 is known to be immunosuppressive
rather than proinflammatory in mammals, its role in
the immune response to A. salmonicida and the pathol-
ogy of furunculosis will be interesting to elucidate.
Of the four cell lines examined, the monocyte ⁄ macro-
phage-like RTS-11 cell line expressed the lowest level of
IL-11 mRNA. However, expression of IL-11 was highly
modulated by stimulation with LPS, bacteria,
poly(I:C), as well as trout recombinant IL-1b in this
cell line. Expression of mammalian IL-11 is modulated
in vitro by cytokines and other stimulants in different
biological settings. IL-1a induces IL-11 expression in
rheumatoid synovial fibroblasts, lung epithelial cell
lines and endometrial epithelial and stromal cells.
TGF-b has been shown to stimulate IL-11 production
in a number of cell types, including lung epithelial cells,
fibroblasts, osteoblasts, chondrocytes, breast cancer
cells, and again endometrial epithelial and stromal cells
[31]. Lastly, with intestinal myofibroblasts, IL-b and

other cell lines.
Bacterial challenge, SSH library construction
and analysis
A virulent strain of A. salmonicida ssp. salmonicida,MT
423 [36,37] was used to challenge rainbow trout ( 300 g,
females) – procedures were in accordance with a Home
Office (UK) animal licence. The preparation of bacteria,
challenge procedure, tissue sampling and construction of
liver SSH libraries were described previously [22]. Fish were
anaesthetized in benzocaine solution (40 mgÆL
)1
) and killed
by severing the spinal cord.
Sequence analysis
The nucleotide sequences generated were assembled and
analysed using the alignir program (LI-COR, Inc., Lin-
coln, NE, USA). A sequence similarity search was per-
formed using fasta [38] and blast [39,40]. Direct
comparison between two sequences was performed using
Rainbow trout interleukin-11 T. Wang et al.
1144 FEBS Journal 272 (2005) 1136–1147 ª 2005 FEBS
the gap program [41]. A signal peptide was predicted using
signalp 3.0 [23]. Multiple sequence alignments of IL-11
molecules from were generated using clustal w, version
1.7 [42]. The IL-11 sequences analysed were from human
[43], monkey (Macaca fascicularis) [1], mouse [44], rat [29]
and fugu (Tetraodon nigroviridis) [45]. Phylogenetic trees of
the IL-6 family members were created from multiple align-
ments by the neighbor-joining method and were boot-
strapped 1000 times.

duct size of 271 bp). One well was identified containing IL-11
cDNA, with an insert of 3 kb, as estimated by PCR using
gene-specific and vector primers. Bacteria from the IL-11-
positive well were diluted in Luria–Bertani medium, incuba-
ted at 37 °C overnight, and re-screened. Finally, the bacteria
were plated, and an isolated clone containing IL-11 was iden-
tified. Plasmid DNA containing the 3 kb IL-11 cDNA was
prepared and fully sequenced from two directions using a
GeneJumper kit (Invitrogen).
Isolation and sequencing of a genomic clone
of IL-11
A rainbow trout genomic library constructed with Lambda
GEM-11 was PCR screened with IL-11-specific primer as
described previously [46]. A positive lambda clone was pla-
que purified and its DNA was prepared using a Wizard
Lambda Preps DNA purification system (Promega). After
an initial restriction enzyme analysis with BamHI, EcoRI,
SacI, XbaI and XhoI, the SacI digestion was subcloned in
pGEM 7zf(+) and sequenced. The cDNA sequence was
aligned to the genomic sequence and the intron ⁄ exon
boundary was identified using the sim4 program (http://
www.hgmp.mrc.ac.uk/Registered/Webapp/sim4/).
In vivo expression of IL-11
A range of tissues (head kidney, gills, liver, spleen, muscle,
skin, intestine and brain) were collected from three healthy
fish for analysis of constitutive expression of IL-11. Liver,
head kidney and spleen tissues were sampled 6, 24 and 48 h
after A. salmonicida MT423 injection as described previ-
ously [22]. Total RNA was prepared using Trizol (Invitro-
gen) and converted to cDNA using PowerScript reverse

Northern blot
Northern blot analysis was performed as described previ-
ously [48]. Briefly, 20 lg of total RNA per lane was trans-
ferred from a 1.1% formaldehyde–Mops agarose gel to
nylon membranes by capillary action and hybridized over-
night at 65 °C with a
32
P-labelled 271 bp cDNA probe
purified from a trout IL-11 PCR fragment amplified from
the IL-11 cDNA clone. A
32
P-labelled b-actin cDNA probe
was used as a control to ensure that any changes in mRNA
levels were not a result of a general change in the amount
of mRNA loaded. Following stringent washing, membranes
were put into an X-ray cassette with intensifying screens
and film (Kodak, Rochester, NY, USA) and exposed for
between 4 h and 4 days.
T. Wang et al. Rainbow trout interleukin-11
FEBS Journal 272 (2005) 1136–1147 ª 2005 FEBS 1145
Acknowledgements
This work was supported by a grant from the EC
(Q5RS-2001–002211). Many thanks to Dr Jun Zou
(University of Aberdeen) for supplying the recombin-
ant trout IL-1b.
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