Báo cáo khoa học: Cloning and expression of the first nonmammalian interleukin-11 gene in rainbow trout Oncorhynchus mykiss pdf

Cloning and expression of the first nonmammalian
interleukin-11 gene in rainbow trout Oncorhynchus mykiss
Tiehui Wang,1 Jason W. Holland,1 Niels Bols2 and Christopher J. Secombes1
1 Scottish Fish Immunology Research Centre, University of Aberdeen, UK
2 Department of Biology, University of Waterloo, Ontario, Canada

Keywords
Aeromonas salmonicida; cloning;
expression; interleukin-11; rainbow trout
Correspondence
C. J. Secombes, Scottish Fish Immunology
Research Centre, School of Biological
Sciences, University of Aberdeen, Aberdeen
AB24 2TZ, UK
Fax: +44 1224 272396
Tel: +44 1224 272872
E-mail:
Database
The nucleotide sequences of rainbow trout
interleukin-11 will appear in the EMBL ⁄
DDBJ ⁄ GenBank nucleotide sequence database under the accession numbers
AJ535687 (cDNA) and AJ867256 (genomic
DNA).
(Received 8 September 2004, revised 9
December 2004, accepted 24 December
2004)
doi:10.1111/j.1742-4658.2005.04544.x

Interleukin (IL)-11 is a multifunctional cytokine that stimulates hematopoietic progenitor cells and exerts a series of important immunomodulatory
effects. It was believed to be restricted to mammals, but here we report the
first nonmammalian IL-11 gene, in rainbow trout (Oncorhynchus mykiss).

A trout IL-11 cDNA clone was isolated that contains a 5¢-untranslated
region (UTR) of 400 bp, an open reading frame of 612 bp and a large 3¢UTR of 1924 bp. Analysis of a genomic DNA clone from a trout lambda
library revealed that the trout IL-11 gene has the same five exon ⁄ four
intron gene organization, as well as the same intron phase, as mammalian
IL-11 genes. The 204 amino acid trout IL-11 translation has a predicted
signal peptide of 26 amino acids and mature peptide of 178 amino acids,
with a calculated molecular mass of 20.5 kDa and a theoretical pI of 9.83.
The mature peptide contains a cysteine residue and a potential N-linked
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.

Interleukin (IL)-11 is a 19 kDa, highly conserved, nonglycosylated 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 precursor and secreted as a 178 amino acid mature peptide 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

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.

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T. Wang et al.

neurotrophic growth factor and a viral homologue of
IL-6 encoded by the Kaposi’s sarcoma-associated herpesvirus. All of these cytokines elicit either hetero- or
homodimerization of gp130, which activates intracellular 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
the body. Basal and inducible IL-11 mRNA expression can be detected in fibroblasts, epithelial cells,
chondrocytes, synoviocytes, keratinocytes, endothelial
cells, osteoblasts and certain tumour cells and cell

lines [3]. Viral [6] and bacterial [7] infection, and
cytokine [IL-1, tumour necrosis factor (TNF)a, and
transforming growth factor (TGF)-b1] [8] stimulation
all induce IL-11 expression. By alternative use of two
polyadenylation sites, the human IL-11 gene produces
two transcripts of 1.5 and 2.5 kb, respectively that
encode the same IL-11 protein [1,9]. Once produced,
IL-11 acts on multiple cell types, including hemotopoietic cells, hepatocytes, adipocytes, intestinal epithelial cells, tumour cells, macrophages, and both
osteoblasts and osteoclasts. In the hematopoietic compartment IL-11 supports multilineage and committed
progenitors contributing to myeloid, erythroid,
megakaryocyte and lymphoid lineages [10]. As human
IL-11 stimulates megakaryocytopoiesis, resulting in
increased production of platelets, it is used in the prevention of severe thrombocytopenia occurring after
cancer chemotherapy and may be a useful future
therapy to ameliorate neonatal thrombocytopenia
[11]. IL-11 is also an anti-inflammatory cytokine that
inhibits the production of proinflammatory cytokines
from lipopolysaccharde (LPS)-stimulated macrophages
[12]. In combination with its trophic effects on the
gastrointestinal epithelium, IL-11 has a profound
activity in the protection and restoration of the
gastrointestinal mucosa [13,14]. Interestingly IL-11
stimulates osteoclast formation and bone resorption
in vitro [15]. Indeed, transgenic overexpression of
IL-11 stimulates osteoblastogenesis and bone formation [16]. Thus, IL-11, together with other members
of the gp130 family, are essential for bone metabolism [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].
FEBS Journal 272 (2005) 1136–1147 ª 2005 FEBS

Rainbow trout interleukin-11

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
Cloning and characterization of rainbow
trout IL-11
A cDNA clone containing a 2936 bp insert has been
isolated from a liver SMART cDNA library and fully
sequenced from both directions. It contained a 5¢-untranslated region (UTR) of 400 bp, an open reading

frame (ORF) of 612 bp and a large 3¢-UTR of
1922 bp (accession number AJ535687, Fig. 1). Several
mRNA instability motifs (ATTTA) were present in the
3¢-UTR as well as in the 5¢-UTR, and four potential
poly(A) signals were found in the 3¢-UTR (Fig. 1), two
of them just 14 or 23 bp upstream of the poly(A) tail.
The remaining two poly(A) signals were upstream of
the region that contained the ATTTA motifs, such that
if either was used as a poly(A) signal, a transcript
encoding the same protein but without any ATTTA
motifs in the 3¢-UTR would be produced. The precursor trout IL-11 translation had 204 amino acids, a calculated molecular mass of 23.3 kDa and a theoretical
isoelectric point (pI) of 9.77. A signal peptide of 26
amino acids was predicted [32]. Thus the mature trout
IL-11 generated following cleavage of the signal peptide is 178 amino acids with a calculated molecular
mass of 20.5 kDa and a theoretical pI of 9.83. A
potential N-linked glycosylation site was also identified
(Fig. 1).
To define the gene organization of trout IL-11, a
lambda clone was isolated and a fragment (9894 bp,
accession number AJ867256) containing the IL-11 gene
and its 5¢- and 3¢-flanking region was sequenced. The
trout IL-11 gene has a five exon ⁄ four intron organization and all introns are phase 0 except intron I, which
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T. Wang et al.

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T. Wang et al.

Rainbow trout interleukin-11

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¢- and 3¢-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¢- and 3¢-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.

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 differences in both the 5¢- and 3¢-UTR. The major differences 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) database 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
coding region with fugu IL-11, compared with 42.5,
43.2, 41.4 and 42.8% with IL-11s from human, monkey, mouse and rat, respectively. Fugu IL-11 translation also showed low homology (28% identity) with
mammalian IL-11s (Table 1). Trout IL-11 has a
mature peptide of the same length (178 amino acids)
as in mammals, although it encodes a longer precursor of 204 amino acids (compared with 199 amino
acids in mammals, Table 2). Both fish IL-11s have a
longer signal peptide of 26 amino acids, compared

Table 1. Amino acid homology of trout IL-11 with known IL-11 molecules.
Puffer fish

Human

Monkey

Mouse

Rat

Identity(%)
Trout

Puffer fish
Human
Monkey
Mouse

Similarity(%)

Identity

Similarity

Identity

Similarity

Identity

Similarity

Identity

Similarity

30.3
–
–
–
–

37.9

–
–
–
–

32.3
28.1
–
–
–

40.3
32.4
–
–
–

31.7
28.6
94.5
–
–

39.8
33.0
95.0
–
–

29.4

28.3
87.9
83.9
–

38.5
32.6
90.0
86.4
–

29.0
27.8
87.9
84.4
97.5

38.5
32.1
90.0
86.9
98.0

Table 2. Comparison of biochemical parameters of the predicted trout IL-11 translation with other known IL-11 molecules.
Puffer fish
Precursor (amino acids)
Signal peptite (amino acids)
Mature peptide (amino acids)
Mature peptide
Mol. mass (kDa)

Cysteine (amino acids)
Potential N-Gly
Prolinea (%)
Leucinea (%)
Positive chargeda (%)
Isoelectric point
a

Trout

Human

Monkey

Mouse

Rat

208
26
182

204
26
178

199
21
178

199
21
178

199
21
178

199
21
178

20.4
4
2
4.9 (9)
15.4 (28)
9.9 (18)
7.91

20.5
1
1
9.0 (16)
13.5 (24)
12.4 (22)
9.83

19.1
0

0
12.4 (22)
23.0 (41)
11.8 (21)
11.16

19.4
0
0
11.2 (20)
23.6 (42)
11.8 (21)
10.71

19.2
0
0
9.0 (16)
21.9 (39)
11.8 (21)
11.16

19.2
0
0
9.0 (16)
21.3 (38)
11.8 (21)
11.16

Percentage of amino acid in the mature peptide. Number in brackets is actual number of residues present.

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Rainbow trout interleukin-11

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
(Table 2).
A multiple alignment was constructed from the fish
(trout and fugu) and mammalian IL-11 molecules
(Fig. 2). The single cysteine residue present in the trout
sequence aligned well with one of the four cysteine residues in the fugu mature peptide. Critical residues L67
and R169 (numbered according to human IL-11) in
the receptor-binding site I, which bind to mammalian
IL-11R and are essential for its activity [2, 24] were
well conserved. However, three residues, K42, M59
and K99, which are conserved in all mammalian
sequences and also critical for receptor binding [5], are

absent in the relevant positions in both fish sequences
(Fig. 2). To define the relationship of trout IL-11 to
other IL-6 family members, a phylogenetic tree was
constructed using the neighbor-joining method and
bootstrapped 1000 times. This tree (Fig. 3) clearly
grouped the trout IL-11 with IL-11 molecules from

T. Wang et al.

other species and separate from other members of the
IL-6 family.
In vivo expression of IL-11
RT–PCR was used to examine the expression of trout
IL-11 in tissues prepared from three healthy fish. Comparable products for b-actin were amplified from the
different samples using a low cycle number (21 cycles).
Trout IL-11 was highly expressed in intestine and gills,
and was also detectable in other tissues including
spleen, liver, head kidney, brain, skin and muscle using
a high cycle number of 32 (Fig. 3). IL-11 expression
was highly up-regulated in vivo by bacterial infection,
as shown in Fig. 4 in liver, head kidney and spleen tissues. No IL-11 expression was detectable using 28
cycles in samples prepared from control fish or phosphate-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 residues (: or.) identified using CLUSTAL W are
indicated. The signal peptides are in bold.
The potential N-glycosylation sites and cysteine residues present in the two fish molecules, and the three conserved residues
(K42, M59 and K99) in the mammalian
mature IL-11 protein, are in bold and underlined. 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.

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T. Wang et al.

Rainbow trout interleukin-11

OSM, Mouse, P53347
OSM, Cow, P53346
OSM, Human, P13725
IL-6, Squirrel Monkey, Q8MKH0
IL-6, Night monkey, Q9TTH3
IL-6, Human. P05231

973
IL-6, Sooty mangabey, P46650
980
IL-6, Rhesus macaque, P51494
966
IL-6, Cynomolgus monkey, P79341
IL-6, Sheep, P29455
984
IL-6, Goat, Q28319
984
IL-6, Cow, P26892
983
IL-6, Buffalo, Q6V919
I L- 6 , K ille r w ha l e, Q 28 474
824
IL-6, Beluga whale, Q9XT80
IL-6, Pig, P26893
952

984

550
984

262

339

450

520

IL-6, Camel, Q865W7
IL-6, Lama glama, Q865X6
IL-6, Cat, P41683
860
IL-6, Dog , P41323
650
IL-6, Seal, Q28819
928
IL-6, Sea Otter, Q28403
IL-6, Woodchuck,
557
IL-6, Rat, P20607
984
IL-6, Mouse, P08505
IL-6, Rabbit, Q9MZR1
499
IL-6, Chicken, Q90YI0
M16, Carp, AY102632
319
IL-6, fugu, Q6L6X6

361

984

971
487

347

353

IL-11, Rat, Q99MF5
IL-11, Mouse, P47873
IL-11, Human, P20809
583
IL-11, Cynomolgus monkey,P20808

984
984
492

IL-11, Trout, AJ535687

412

IL-11, Fugu, Q6UAM0
809
IL-11, Halibut, AU090873 (est)
702
IL-11, Cat fish, CB939978 (est)
LIF, Possum, Q9GLC2
LI F , P ig , Q 9 G K Z 8
341
LIF, Mouse, P09056
984
LIF, Rat, P17777
LIF, Human, P15018

466
LI F , S h e e p , Q 7 M3 6 3
259
LIF, Mink, O62728
175
LIF, Cow, Q27956
CT-1, Human, Q16619
984
CT-1, Rat ,Q63086
984
CT-1, Mouse, Q60753

403

984

984
984

636

984
984

840

726
805

CNTF, Chicken, Q02011

CNTF, Pig, O02732
CNTF, Rat, P20294
CNTF, Mouse, P51642
CNTF, Human, P26441
CNTF, Rabbit, P1 4188

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 factor), OSM (Oncostatin M), CT-1 (cardiotrophin-1) and CNTF (ciliary neurotrophic factor). A carp molecule (M16) that shares sequence similarity with the IL-6 family is also included. The tree was constructed using the neighbor-joining method. Numbers indicate bootstrap values
from 1000 replications. The molecular type, species and accession number for each molecule analysed are on the right.

cultures one day after passaging. Expression of IL-11
was highly detectable in CL-6 and RTG cell lines, and
to a lesser extent in RTL cells. However, only a very
weak PCR product was detectable in the monocyte ⁄ macrophage cell line RTS-11, under 32 cycles of
amplification (Fig. 5). As a potentially important molecule in monocyte ⁄ macrophage host defence, the modulation of IL-11 was studied in RTS-11 cells. One day
after passaging, RTS-11 cells were stimulated with
LPS (25 lgỈmL)1), Aeromonas salmonicida MT423
[multiplicity of infection (MOI) ẳ 50] or poly(I:C)
(25 lgặmL)1) for 3, 7 and 24 h. IL-11 expression in
FEBS Journal 272 (2005) 1136–1147 ª 2005 FEBS

RTS-11 cells was highly up-regulated by bacterial
infection, as well as by LPS, poly(I:C) (Fig. 6A) and
recombinant trout IL-1b stimulation (Fig. 6B).
Northern blot analysis
To study the transcript size and to confirm the RT–
PCR analysis, RNA samples from LPS (25 lgỈmL)1,
4 h) and trout recombinant IL-1b (25 ngỈmL)1, 4 h)
stimulated RTS-11 cells were analysed by northern
blotting. A single transcript of  3.2 kb was detectable
that was highly expressed in LPS- and recombinant

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3

2

3 1

1

2

3 1

2

3

2

3

1

2

3

1

2

3

pM
ar
k

lee
n
Sp
1

er

id
ne
y
3

Li
ve
r

le
M
us
c

in
Sk
2

He
ad
k

e
tin
In
tes
1

0b

2

Gi
lls

1

T. Wang et al.

10

10

0b

Ne p M
ga ar
tiv ke
ec r
o
Br
ain ntro
l

Rainbow trout interleukin-11

β

IL-11

PBS

Bacterial

Liver

D

NA
m
6 ar
h ke
r
24
h

48
h
6
h
24
h
48
h
C
on
N trol
eg
.
D con
NA tr
m ol
ar
ke
r

Fig. 4. Expression of trout IL-11 in tissues. A range of cDNAs was prepared from tissue samples from three healthy rainbow trout and used
for amplification of IL-11 (Primer EF and ER, expected size: 271 bp) and b-actin (expected size: 276 bp). The cycle numbers were 21 and 32
for b-actin and IL-11, respectively.

β-Actin

Spleen

Head kidney

IL-11
β-Actin

IL-11

β-Actin

IL-11

IL-1b-treated samples but undetectable in the control
cells. Comparable signals for the 1.8-kb b-actin transcript were detected in all samples (Fig. 7A,B).

Discussion
This is the first report to describe an IL-11 gene in
fish. The trout IL-11 gene was initially identified from
1142

Fig. 5. Expression of trout IL-11 in vivo in
bacterially challenged rainbow trout. Trout
were injected with PBS or bacteria (Aeromonas salmonicida, MT423) and three fish
were sampled at 6, 24 and 48 h postinjection, as described in the Experimental procedures. 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.

a liver SSH library prepared from bacterial-challenged
fish and a full-length cDNA clone was then obtained
from a SMART cDNA library. The trout sequence
has a long ORF encoding a basic protein with a predicted signal peptide of 26 amino acids and a mature

peptide of 178 amino acids. The trout IL-11 translation, as well as one from another bony fish (fugu,
Tetraodon nigroviridis) share low amino acid identity
FEBS Journal 272 (2005) 1136–1147 ª 2005 FEBS


24 h

7h

Poly I:C
3h

24 h

7h

3h

MT423
24 h

7h

LPS
3h

Control

Marker

A

Marker

Rainbow trout interleukin-11

Neg. Control

T. Wang et al.

β-actin

LP
S

C

on

tr
ol

A

β-actin

C

on

β
rI

L
-1

β
-1
L

tr
ol

1.8 kb

rI

Marker

8h

6h

4h

2h

IL-11

IL-11

3.2 kb

IL-11

1.8 kb

β-actin

Fig. 7. Northern blot analysis of trout IL-11 expression in RTS-11
cells stimulated for 4 h with LPS (25 lgỈmL)1) (A) and recombinant
trout IL-1b (25 ngỈmL)1) (B). Twenty micrograms of RNA was separated, transferred to a Hybond nylon membrane, and hybridized
with 32P-labelled trout b-actin and IL-11 probes. Two independent
experiments are shown for the LPS or rIL-1b stimulated cells.

(28–32%) to mammalian IL-11, despite the fact that
mammalian IL-11s are highly conserved and share
94% amino acid identity between primate sequences,
97% identity between mouse and rat sequences, and
88% identity between human and mouse sequences.
FEBS Journal 272 (2005) 1136–1147 ª 2005 FEBS

1h

β-actin

3.2 kb

B

0.5 h

0h

Marker

B

LP
S

Fig. 6. Modulation of IL-11 expression by
LPS, bacterial infection and Poly I:C (A) or
trout rIL-1b (B). One day after passaging,
RTS-11 cells were treated with LPS
(25 lgặmL)1), Aeromonas salmonicida
MT423 (MOI ẳ 50), or poly(I:C) (50 lgỈmL)1)
for 3, 7 and 24 h or IL-1b (25 ngỈmL)1) for
0.5–8 h, and total RNA prepared for
RT–PCR analysis. The cycle numbers for
PCR were 21 and 30 for b-actin and IL-11,
respectively.

Neg. Control

IL- 11

The trout molecule has a similar exon ⁄ intron organization to mammalian IL-11. The phylogenetic tree
grouped the trout molecule with IL-11 molecules from
other species and separate from other IL-6 family

members. Such evidence strongly suggests that the
trout molecule is the fish homologue to mammalian
IL-11. The low homology between fish and mammalian sequences is not unusual. For example, trout
IL-1b shares 28% identity with human IL-1b [25],
trout TNFa shares 32% identity with human TNFa
[26], and a recently identified trout IL-15 shares only
26% identity with human IL-15 [27]. The divergence
between fish and mammalian IL-11 and the conservation between mammalian IL-11 molecules may be relevant to their functional role. For example, the
requirement for IL-11 in normal development of placentation in mammals is a biological activity that is
apparently not needed in fish. However, other functions, 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-transcription 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
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Rainbow trout interleukin-11

protein [1], and respond similarly to IL-1a ⁄ b and TGFb stimulation [8,28]. In mouse and rat only a single IL11 transcript, of 2 kb, is produced [29]. The trout IL-11
gene gives rise to a single transcript of 3.2 kb in RTS
cells, as seen in the northern blot (Fig. 7), and is the
largest of the known IL-11 molecules. Interestingly, the
trout IL-11 transcript has four potential poly(A) signals

(AATAAA), two of them present just 14 and 23 bp,
respectively, upstream of the poly(A) tail in the cDNA
sequence, which should be the poly(A) signal giving rise
to the 3.2 kb transcript. The other two are present
before the region that contains the ATTTA motifs and
if either is used as a poly(A) signal, a transcript encoding the same protein but without instability motifs in
the 3¢-UTR would be produced, as a shorter but potentially more stable mRNA.
IL-11 expression was detectable by RT–PCR in all
the tissues examined, with apparent high levels of
expression in intestine and gills. Intestine and gills are
two primary sites where fish encounter foreign insults
including infection by bacteria, viruses and parasites,
and environmental stresses. Constitutive expression of
IL-11 was also detectable in lung, stomach and intestine in mice [30], and suggests a conserved role for
IL-11 in gills ⁄ lung and intestine, where it functions as
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 bacterial infection. This induction of expression has also
been seen after infection in mammals where, for example, human cord blood-derived macrophages and dendritic 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 aeruginosa [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 pathology of furunculosis will be interesting to elucidate.
Of the four cell lines examined, the monocyte ⁄ macrophage-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
1144

T. Wang et al.

cells, and again endometrial epithelial and stromal cells
[31]. Lastly, with intestinal myofibroblasts, IL-b and
TGF-b are potent inducers of IL-11 expression at both
the mRNA and protein levels [8]. Trout RTS-11 cells
show constitutive expression of TGF-b mRNA, and
IL-1b but not TGF-b mRNA is up-regulated by LPS
and IL-1b itself [32]. IL-1b mRNA is also up-regulated
in RTS-11 cells by infection with A. salmonicida and by
poly(I:C) stimulation (unpublished results). Thus, the
up-regulation of IL-11 expression by such stimuli may
be an indirect effect through the induction of IL-1b.
In conclusion, it is clear that IL-11 evolved early
and is present in bony fish. Expression of IL-11 in
trout was shown to be widely distributed, being present in all the tissues and cell lines examined. Its
expression was modulated by bacterial infection, and
stimulation in vitro with LPS, poly(I:C) (a viral mimic)
and IL-1b. Such data suggest that IL-11 is an active
player in the cytokine network and the host immune
response to infection in fish.

Experimental procedures
Cell lines and cell culture
Four rainbow trout cell lines were used in these studies; the
mononuclear cell line RTS-11 [33], the gonad cell line RTG
[34], the liver cell line RTL [35] and CL-6 (a generous gift
from Dr Benmansour, INRA, France). All trout cells were
grown in L-15 medium supplemented with 100 unitsỈmL)1
penicillin and 100 lgỈmL)1 streptomycin, and 30% fetal calf
serum for RTS-11, and 10% fetal bovine serum for all the
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 mgỈL)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., Lincoln, NE, USA). A sequence similarity search was performed using fasta [38] and blast [39,40]. Direct
comparison between two sequences was performed using

FEBS Journal 272 (2005) 1136–1147 ª 2005 FEBS

T. Wang et al.

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 alignments by the neighbor-joining method and were bootstrapped 1000 times.

Trout IL-11 cDNA cloning
To clone full-length trout IL-11 cDNA, a SMART cDNA
library was constructed from bacterial-challenged liver tissues, as high expression of IL-11 was seen after bacterial
infection in initial RT–PCRs. Briefly, SMART cDNA was
synthesized using total RNA prepared from liver tissue 24
and 48 h postchallenge with A. salmonicida MT423, using a
SMART cDNA synthesis kit (Clontech, Hampshire, UK).
The SMART cDNA was amplified over five cycles using the
Advantage-2 PCR enzyme system (Clontech) and a modified
primer (TGAAAGCGGCCGCAGTGGTATCAACGCAG
AGT) with a NotI restriction enzyme site at the 5¢-end. The
resulting amplified cDNA was digested with NotI (Promega,
Southampton, UK), purified using a Qiagen PCR purification kit (Qiagen, West Sussex, UK) and ligated to NotI
digested, dephosphorylated pGEM 5Zf(+) (Promega).
Competent cells UltraMaxTM DN5a-FTTM (Invitrogen, Paisley, UK) were transformed with the ligated cDNA, diluted in
200 mL Luria–Bertani medium, containing 100 lgỈmL)1
ampicillin, after 1 h recovery incubation at 37 °C and dispersed into two 96-deep-well (2.2 mL) plates and incubated
at 37 °C overnight. The bacteria from each well were PCR
screened with IL-11 specific primers (Forward primer, 5¢TCAACTCCCTTGAGATGAGACC-3¢; Reverse primers,

5¢-TCCTGGGAAGACTGTAACACATC-3¢, expected product 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-11positive well were diluted in Luria–Bertani medium, incubated at 37 °C overnight, and re-screened. Finally, the bacteria
were plated, and an isolated clone containing IL-11 was identified. 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 plaque purified and its DNA was prepared using a Wizard
Lambda Preps DNA purification system (Promega). After
an initial restriction enzyme analysis with BamHI, EcoRI,

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Rainbow trout interleukin-11

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 previously [22]. Total RNA was prepared using Trizol (Invitrogen) and converted to cDNA using PowerScript reverse
transcriptase (Clontech). PCR was performed with IL-11
gene-specific primers as described above for cloning of the
IL-11 cDNA. A parallel PCR using primers for b-actin
(forward primer, 5¢-CGACCTCACAGACTACCTGAT-3¢;
reverse primers, 5¢-TGGATACCGCAAGACTCCATAC-3¢,
expected product size of 276 bp) was used as a positive control for RT-PCR. The PCR conditions were as described
previously [46].

Modulation of IL-11 expression in cell lines
One day after passaging, cells were used for preparation of
total RNA to examine constitutive expression of IL-11. To
study the modulation of IL-11 expression in RTS-11 cells,
one day after passaging cells were treated with 25 lgỈmL)1
LPS (Sigma), live A. salmonicida MT423 (MOI ẳ 50) or
50 lgặmL)1. Poly(I:C) (Sigma, Dorset, UK) for 3, 7 and
24 h, and recombinant trout IL-1b [47] (25 ngỈmL)1) for
0.5–8 h. Total RNA was prepared from the treated and
untreated control cells and analysed by RT–PCR as described above. Total RNA was also prepared from RTS-11
cells treated with LPS (25 lgỈmL)1) and recombinant trout
IL-1b (25 ngỈmL)1) for 4 h for northern blot analysis.

Northern blot
Northern blot analysis was performed as described previously [48]. Briefly, 20 lg of total RNA per lane was transferred from a 1.1% formaldehyde–Mops agarose gel to
nylon membranes by capillary action and hybridized overnight at 65 °C with a 32P-labelled 271 bp cDNA probe
purified from a trout IL-11 PCR fragment amplified from
the IL-11 cDNA clone. A 32P-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.

1145


Rainbow trout interleukin-11

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 recombinant trout IL-1b.

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