AbstractUsing PCR and Northern blot analysis, an IL-1 receptor antagonist specific transcript was amplified from HepG2- and liver mRNA, cDNA clones coding for IL-1 receptor antagonist were isolated from a liver cDNA library and sequence comparison revealed complete identity with the secreted, monocytic form of IL-1 receptor antagonist

Estaller, C.

Sim, Robert B.

Steinkasserer, Alexander

Weiss, Elisabeth H.

English

Universität München: Elektronischen Publikationen

Volume 310, number 1, 60-62 FEBS 11476 
© 1992 Federation of European Biochemicai Societies 00145793/92/$5.00 
September 1992 
Human interleukin-1 receptor antagonist is expressed in liver 
Alexander  Steinkasserer~'~ Corne l ia  Estaller b, El isabeth H. Weiss b and  Robe r t  B. Sim:' 
"MRC bnmunochemistr), Unit, Department of  Biochemistr.v, University of O.xford, South Parks Road, O.xford, OXI 3QU, UK and 
t'1nstitute of bnmtmology, University of Munich, Goethestr. 31, 8000 M~nich.2. German t, 
Received 14 July 1992 
Using PCR and Northern blot analysis, an IL-I receptor antagonist specific transcript was amplified from HepG2- and liver mRNA. eDNA clones 
codln8 for IL- 1 receptor antagonist were isolated from a liver eDNA library and sequence comparison revealed complete identity with the secreted, 
monocytie form of IL-1 receptor antagonist. 
IL-I receptor antagonist; Liver: eDNA transcript 
1. INTRODUCTION 
IL-I receptor antagonist (IL-lra) is a protein which 
inhibits IL-1 (IL-I~ and IL-lfl) activity, by binding 
competitively to IL-I receptors [1]. This naturally oc- 
curring inhibitor has been shown to reduce the severity 
of sepsis, colitis, arthritis and diabetes in animals and 
it therefore plays an important role in the control of 
IL-I mediated inflammatory and autoimmune diseases 
[2]. IL-lra is structurally related to IL-1 and shows 18% 
and 26% identity in amino acid sequence to IL-I~ and 
li.-lfl, respectively. 
The eDNA sequence of IL-lra derived from mon- 
ocytes has been previously determined [3] and using an 
intronic polymorphism [4] the localization of the gone 
on chromosome 2 has been established by linkage anal- 
ysis [5], as well as by in situ hybridisation [6]. 
There are two types of IL-lra, one secreted, soluble 
form expressed in monocytes and netttrophils and an 
intracellular form expressed in epithelial cells. This in- 
tracellular IL-lra, which lacks a leader peptide, is de- 
rived from the same gene through the use of an alterna- 
tive transcriptional start site and alternative splicing [7]. 
In this study we report the expression of the secreted 
form of the lL-lra in liver. 
2. MATERIALS AND METHODS 
2.1. eDNA ~ynthesis and PCR amplification 
Total RNA was isolated from the human cell lines HepG2, Chang, 
U937, Daudi, HL60, Molt4, Raji and T47D as well as from liver, 
temporal cortex and lung tissue using guanidinium thiocyanate and 
a CsCI gradient [8]. 10/zg RNA was used for the first strand eDNA 
synthesis (eDNA synthesis kit; Amersham, Bucks, UK) and 1/10tb of 
this reaction mixture was used for PCR amplification. Th~ two PCR 
Correspondence address: A. Steinkasserer, MRC Immunoehemistry 
Unit, Dept. Biochemistry, South Parks Road, Oxford, OXI 3QU, 
UK. Fax: (44) (865) 275729 
primer sets described by Haskill et al. [7] were used !n order to distin- 
guish between RNA transcripts for he secreted and the intracellular 
forms of t L-Ira. PCR products were analysed after 30 cycles (94°C, 
1 min; 60"C, 30 s, 72°C, 2 rain) on a 0.7% (w/v) agarose gel. 
2.2. Northern blot ana6,~'is 
A 10/.tg portion of total RNA was electrophoresed in 1.2% (w/v) 
agarose gel and the RNA transferred to a Hybond-N membrane 
(Amersham) and hybridised at 65°C with a radiolabelled IL-lra probe 
dei'ived fi'om HepG2 mRNA b2¢ PCR. The blots were washed at high 
stringency [8]. 
2.3. Isolation of lL-lra clones and eDNA sequencing 
A human liver cDNA library constructed in the eukaryotic expres- 
sion vector CDM8 [9] was screened with an IL-lra probe derived from 
HepG2 mRNA by PCR. The length of the positive cDNA clones was 
estimz ..d by a Pstl.Hindlll restriction digest. The sequence was deter- 
mined from both strands using the double-strand sequencing kit from 
Pharmacia (Milton Keyne~, UK), 
3. RESULTS AND DISCUSSION 
Using specific PCR primers it was possible to amplify 
an IL- Ira transcript from the hepatoma cell line HepG2 
and from total liver RNA. In liver on!y the secreted 
form of the antagonist was present (Table I) and no 
amplification product was obtained for the intracellular 
form. Interestingly no amplification product was ob- 
tained from the Chang liver cell line which is derived 
from nonmalignant human liver tissue, indicating a loss 
of  the IL-Ira message in this cell line or need for an 
external stimulus. As a positive control the monocytic 
cell line U937 was used. The other cell lines (Molt4, 
HL60, Daudi, T47D and Raji) and tissues (temporal 
cortex and lung) were negative for the secreted (Table 
I) as well as for the intracellular IL-lra  (data not 
shown). 
To confirm the PCR analysis, ,Northern blots were 
performed with RNA extracted from liver tissue and 
from the cell lines HepG2, Chang and Raji. As shown 
60 P, blished by Elsevier Science Publishers B. V. 
Volume 310, number 1 FEBS LETTERS September 1992 
I(, 0 I~i H C R 
,~.~'-' ~ . i " . : " . : . " : " ' , : ,  :~ ' . "  ~ - : ~ "  , . 
,...-.:~..:, -:-: : ~  : . : . , " . . . , . 
; . . ' :  .~ ; . . ,  . . . . . .  . : . . - r . , , 2  :: . . . - : : : . . . . ' .  , . . , .  :.. 
i:~i::i:'?: ': " '  : ~: .~;~ '- :  ::/i~::i~' 
;,.i:~;¢:.. ! . . . .  : ":.-.:; ::!~ ? ,::6: / ~ 
~, : ' . c  - " ~.  ' ~  " • " " ,  ~ .  
~Z'-.~.' , : : ? i ~ ; "  '-~ : ,  
~ ~'~": :.~:.... ~ : ":t'~!': ' ~ 
: : : ~ , '  • . : .~ : ; , : :  : .  
~ ' : ,~0 ' . . .  ' . - . . ~ - ,  : - : - . ' : ! . . . ~  " . ,  
.~::::'- -~'~ .. :,~-~ . : ~  
. . . .  . . .  ' ~ . . . . . . .  ; - . . . . . . . . . . .  ,.: 
• '. ; i~77~.: ; ? . ~ . . .  
: - ..~-. • ¢!~ ;~5~;.i f.~ 
. : ..r " : : . . , : : ~ . :  ~ ~::: " , . ~  
• . . . .  • . .. . • . . . . . : ; . , . .  , y  , . ' . . ' , ~ ; / ,  
, ' . i  ~ '  " :  . . . . . . .  . . . .  : - ~  ,i ~: : " ?  ~ "  
" . ' "  ; " - ' . f . ?  • .! i . ( : .  . : " ' . ~ : ~  " , ? i  r~ 
-i,~. ~:,:';,..,, ~ . : : , ~ . . . , , ~  
.~. ,~ ' ; i : : , ; ; ' . - " ;  " ~ : -  ¢ : " : ~ . .  " - "  / . ' . ' 7 £ : - ~ : : ' " ' ? . " .  
I L - I  R A - -  
Fig. 1. Northern blot analysis of total RNA isolated from liver tissue 
(lanes 1-3; individuals: K, O, N,) and the cell lines HepG2 (H), Chang 
(C) and gaji (R) probed with an IL-lra eDNA probe. The additional 
fainter bands are probably due to background-hybridization or not 
totally spliced mRNA. 
in Fig. 1 all three liver RNAs (individuals: K, O, N) 
contained the IL-lra transcript (lanes 1-3), as did the 
HepG2 cell line (lane 4). No IL-I ra transcript was de- 
tected in the cell lines Chang and Raji (lanes 5 and 6), 
in agreement with the findings obtained by PCR analy- 
sis. 
To establish the sequence of  this liver IL-lra tran- 
script a liver cDNA library subeloned into the CDM8 
expression vector was screened with an IL-lra probe. 
Three positive clones were isolated and a 1.65 kb insert 
from clone p lLlra-L was sequenced. This clone con- 
tains a short Y-untranslated region (8 bp) followed by 
Table I 
PCR analysis of cell line and tissue eDNA transcripts 
sec. IL-I ra 
HepG2 
Chang 
Liver 
U937 
Daudi 
HL60 
Molt4 
gaji 
T47D 
Cortex 
Lung 
(hepatoma) + 
(hepatocyte) 
(tissue) + 
(monoeytie) + 
(B.lymphoblastoid) 
(net,trphilic) 
(T-lymphoblastoid) 
(B-lymphobla~toid) 
(breast carcinoma) 
(tissue) 
(tissue) 
the leader sequence and the coding region for the ma- 
ture secreted protein. The T-untranslated region is 1108 
bp long. Sequence comparison revealed that this se- 
quen~ is identical to the eDNA corresponding to the 
secreted form of the 1L-lra from monocytes [3]. 
Since the IL-ra transcript was detected (by PCR and 
Northern blot) in liver tissue as well as in the hepatoma 
cell line HepG2 the transcript in the tissue is likely to 
be derived from hepatocytes and therefore represents a 
true IL-lra transcript in human liver. 
Zahedi et al. demonstrated the induction o f  the IL- 
Ira gene during inflammatory and acute phase reac- 
tions in the mouse liver, suggesting a possible involve- 
ment in the control of inflammatory mediators [10]. 
Many proteins involved in immune and inflammatory 
responses are expressed and/or controlled in the liver by 
an IL-1 stimulus mainly derived from macrophages. 
This includes several complement proteins such as C3, 
C5 and Factor B as well as acute phase proteins such 
as serum amyloid P component [11] and serum amyloid 
A [12]. It is necessary to control this IL-1 transcriptional 
activation in the liver in order to prevent an overexpres- 
sion which could lead to pathophysiological effects. 
Therefore, the existence o f  a natural IL-lra expressed 
in the liver is essential and the knowledge of  the eDNA 
sequence of  this transcript opens the possibility to inves- 
tigate the specific control and the interaction with other 
proteins in the liver environment. The expression of  the 
secreted form of the lL-lra in liver is consistent with the 
fact that this protein competes in the extraeellular envi- 
ronment with [L-I for the binding to IL-I receptors. 
A c k n o w l e d g e m e n t s :  We would like to thank Dr K.B.M. Reid for 
helpful discussion and critisism of the manuscript. "rhi~ work is a 
contribution from the Oxford Centre of Molecular Science (OCMS) 
which is supported by SERC and the MRC. A.S. was supported by 
an OCMS post-doctoral fellowship. We also thank the Yamanouchi 
Research Institute UK for support. 
REFERENCES 
[1] Arend, W,P., Joslin, F.G. and Massoni, R.J. (1985) J. lmmunol. 
134, 3868-3875. 
[2] Dinarello, C.A. and Thompson, R.C, (1991) I mmnnol. Today i 2, 
404--410. 
[3] Eisenber 8, S.P,, Evans, g,J., Arend, W.P., Verderber, E., Brewer, 
M,T,, Haanum, C.H. and Thompson, R.C. (1990) Nature 343, 
341-346. 
[4] Steinkasserer, A., Koelble, K. and $im, R.B. (1991) Nueleie Acids 
Res. 19, 5095. 
[5] Steinkasserer, A., Spurt, N .K,  Cox, S., Jeggo, P. and S i r e ,  R.B. 
(1992) Genomics 13, 654-657, 
[6] Lennard, A,, German, P., Carrier, M., Scotney, H., Sheer, D. and 
Solad, R. (1992) Cylokine 4, 83-89. 
[7] Haskill, S., Martin, G., Van Le, L., Morris, J ,  Peace, A., Bigler. 
C.F., Jafli~, G.J., Hammerberg, C., Spora, S.A., Forte, S., Arend, 
W.P. and Ralph, P. (1991) Prec. Natl, Aead. Sei. U S A  8 8 ,  3681- 
3685. 
[8] Maniatis, T,, Frit~clt, E.F. and Sarnbrook, J. (1982) A Labora- 
tory Manual, Cold Spring Harbor Press, Cold Spring Harbor. 
61 
Volume 310, number 1 FEBS LETTERS September 1992 
[9] Estaller, C., Sehwaeble, W., Dierieh, M.P. and Weiss, E.H. (1991) 
Ear. J. Immunol. 21,799-802. 
[10] Zahedl, K., $eldin, M.F., Rim, M., Ezekowitz, A.B. and White- 
head, A.S. (1991) J. h'araun~l. 146, 4228-4233. 
[11] Zahdi, K. and Whitehead, A.S. (1989) J. lmmunol. 143, 2880- 
2886. 
[12] St¢inkassor~r, A., Weiss, E.H., Schwaebl¢, W. and Link¢, R,P. 
(1990) Biochem. J. 268, 187-193. 
62 


Human interleukin-1 receptor antagonist is expressed in liver

Estaller, Cornelia

Elsevier - Publisher Connector 

Volume 310, number 1, 60-62 FEBS 11476 
© 1992 Federation of European Biochemicai Societies 00145793/92/$5.00 
September 1992 
Human interleukin-1 receptor antagonist is expressed in liver 
Alexander Steinkasserer~'~ Cornel ia Estaller b, El isabeth H. Weiss b and Robert  B. Sim:' 
"MRC bnmunochemistr), Unit, Department of Biochemistr.v, University of O.xford, South Parks Road, O.xford, OXI 3QU, UK and 
t'1nstitute of bnmtmology, University of Munich, Goethestr. 31, 8000 M~nich.2. German t,
Received 14 July 1992 
Using PCR and Northern blot analysis, an IL-I receptor antagonist pecific transcript was amplified from HepG2- and liver mRNA. eDNA clones 
codln8 for IL- 1 receptor antagonist were isolated from a liver eDNA library andsequence omparison revealed complete identity with the secreted, 
monocytie form of IL-1 receptor antagonist. 
IL-I receptor antagonist; Liver: eDNA transcript 
1. INTRODUCTION 
IL-I receptor antagonist (IL-lra) is a protein which 
inhibits IL-1 (IL-I~ and IL-lfl) activity, by binding 
competitively to IL-I receptors [1]. This naturally oc- 
curring inhibitor has been shown to reduce the severity 
of sepsis, colitis, arthritis and diabetes in animals and 
it therefore plays an important role in the control of 
IL-I mediated inflammatory and autoimmune diseases 
[2]. IL-lra is structurally related to IL-1 and shows 18% 
and 26% identity in amino acid sequence to IL-I~ and 
li.-lfl, respectively. 
The eDNA sequence of IL-lra derived from mon- 
ocytes has been previously determined [3] and using an 
intronic polymorphism [4] the localization of the gone 
on chromosome 2 has been established by linkage anal- 
ysis [5], as well as by in situ hybridisation [6]. 
There are two types of IL-lra, one secreted, soluble 
form expressed in monocytes and netttrophils and an 
intracellular form expressed in epithelial cells. This in- 
tracellular IL-lra, which lacks a leader peptide, is de- 
rived from the same gene through the use of an alterna- 
tive transcriptional start site and alternative splicing [7]. 
In this study we report the expression of the secreted 
form of the lL-lra in liver. 
2. MATERIALS AND METHODS 
2.1. eDNA ~ynthesis and PCR amplification 
Total RNA was isolated from the human cell lines HepG2, Chang, 
U937, Daudi, HL60, Molt4, Raji and T47D as well as from liver, 
temporal cortex and lung tissue using guanidinium thiocyanate and 
a CsCI gradient [8]. 10/zg RNA was used for the first rand eDNA 
synthesis (eDNA synthesis kit; Amersham, Bucks, UK) and 1/10tb of 
this reaction mixture was used for PCR amplification. Th~ two PCR 
Correspondence address: A. Steinkasserer, MRC Immunoehemistry 
Unit, Dept. Biochemistry, South Parks Road, Oxford, XI 3QU, 
UK. Fax: (44) (865) 275729 
primer sets described by Haskill et al. [7] were used !n order to distin- 
guish between RNA transcripts for he secreted and the intracellular 
forms of t L-Ira. PCR products were analysed after 30cycles (94°C, 
1 min; 60"C, 30 s, 72°C, 2 rain) on a 0.7% (w/v) agarose gel. 
2.2. Northern blot ana6,~'is 
A 10/.tg portion of total RNA was electrophoresed in 1.2% (w/v) 
agarose gel and the RNA transferred to a Hybond-N membrane 
(Amersham) and hybridised at 65°C with a radiolabelled IL-lra probe 
dei'ived fi'om HepG2 mRNA b2¢ PCR. The blots were washed at high 
stringency [8]. 
2.3. Isolation of lL-lra clones and eDNA sequencing 
A human liver cDNA library constructed in the eukaryotic expres- 
sion vector CDM8 [9] was screened with anIL-lra probe derived from 
HepG2 mRNA by PCR. The length of the positive cDNA clones was 
estimz ..d by a Pstl.Hindlll restriction digest. The sequence was deter- 
mined from both strands using the double-strand sequencing kit from 
Pharmacia (Milton Keyne~, UK), 
3. RESULTS AND DISCUSSION 
Using specific PCR primers it was possible to amplify 
an IL- Ira transcript from the hepatoma cell line HepG2 
and from total liver RNA. In liver on!y the secreted 
form of the antagonist was present (Table I) and no 
amplification product was obtained for the intracellular 
form. Interestingly no amplification product was ob- 
tained from the Chang liver cell line which is derived 
from nonmalignant human liver tissue, indicating a loss 
of the IL-Ira message in this cell line or need for an 
external stimulus. As a positive control the monocytic 
cell line U937 was used. The other cell lines (Molt4, 
HL60, Daudi, T47D and Raji) and tissues (temporal 
cortex and lung) were negative for the secreted (Table 
I) as well as for the intracellular IL-lra (data not 
shown). 
To confirm the PCR analysis, Northern blots were 
performed with RNA extracted from liver tissue and 
from the cell lines HepG2, Chang and Raji. As shown 
60 P, blished by Elsevier Science Publishers B. V. 
Volume 310, number 1 FEBS LETTERS September 1992 
I(, 0 I~i H C R 
,~.~'-' ~ . i " . : " . : . " : " ' , : ,  :~' ."  ~- :~"  , . 
,...-.:~..:, -:-: :~  : . : . , " . . . , . 
; . . ' :  .~ ; . . ,  . . . . . .  . : . . - r . , ,2  :: . . . - : : : . . . . ' .  ,..,. :.. 
i:~i::i:'?: : " '  : ~:.~;~'-: ::/i~::i~' 
;,.i:~;¢:.. ! . . . .  : ":.-.:; ::!~ ? ,::6: / ~ 
~, : ' . c  - " ~. '~  " • " " ,  ~ .  
~Z'-.~.' , : : ? i~ ; "  '-~ :, 
~ ~'~": :.~:.... ~ : ":t'~!':  ~ 
: : :~ , '  • .:.~ : ; , : :  : .  
~':,~0'. . .  ' . - . .~- ,  : - : - . ' : ! . . .~  " . ,  
.~::::'- -~'~ .. :,~-~ . :~  
. . . .  . . .  ' ~ . . . . . . .  ; - . . . . . . . . . . .  ,.: 
• '. ;i~77~.: 7~;? .~. . .  
: - ..~-. • ¢!~ ; 5~;.i f.~ 
. : ..r " : : . . , : :~ . :  ~ ~::: " , . ~  
• . . . .  • . .. . • . . . . . : ; . , . .  , y  , . ' . . ' ,~ ; / ,  
, ' . i  ~ '  " :  . . . . . . .  . . . .  : -~  ,i ~: : "?  ~"  
" . ' "  ; " - ' . f .?  • .! i .(:. . : " ' .~ :~ " , ? i  r~ 
-i,~. ~:,:';,..,, ~ . : : ,~ . . . , ,~  
.~.,~ ' ; i : : , ; ; ' . - " ;  "~: -  ¢ : " :~. .  " - "  / . ' . ' 7£ : -~: : ' " ' ? . " .  
I L - I  RA- -  
Fig. 1. Northern blot analysis of total RNA isolated from liver tissue 
(lanes 1-3; individuals: K, O, N,) and the cell lines HepG2 (H), Chang 
(C) and gaji (R) probed with an IL-lra eDNA probe. The additional 
fainter bands are probably due to background-hybridization or ot 
totally spliced mRNA. 
in Fig. 1 all three liver RNAs (individuals: K, O, N) 
contained the IL-lra transcript (lanes 1-3), as did the 
HepG2 cell line (lane 4). No IL-I ra transcript was de- 
tected in the cell lines Chang and Raji (lanes 5 and 6), 
in agreement with the findings obtained by PCR analy- 
sis. 
To establish the sequence of this liver IL-lra tran- 
script a liver cDNA library subeloned into the CDM8 
expression vector was screened with an IL-lra probe. 
Three positive clones were isolated and a 1.65 kb insert 
from clone plLlra-L was sequenced. This clone con- 
tains a short Y-untranslated region (8 bp) followed by 
Table I 
PCR analysis of cell line and tissue eDNA transcripts 
sec. IL-I ra 
HepG2 
Chang 
Liver 
U937 
Daudi 
HL60 
Molt4 
gaji 
T47D 
Cortex 
Lung 
(hepatoma) + 
(hepatocyte) 
(tissue) + 
(monoeytie) + 
(B.lymphoblastoid) 
(net,trphilic) 
(T-lymphoblastoid) 
(B-lymphobla~toid) 
(breast carcinoma) 
(tissue) 
(tissue) 
the leader sequence and the coding region for the ma- 
ture secreted protein. The T-untranslated region is 1108 
bp long. Sequence comparison revealed that this se- 
quen~ is identical to the eDNA corresponding to the 
secreted form of the 1L-lra from monocytes [3]. 
Since the IL-ra transcript was detected (by PCR and 
Northern blot) in liver tissue as well as in the hepatoma 
cell line HepG2 the transcript in the tissue is likely to 
be derived from hepatocytes and therefore represents a
true IL-lra transcript in human liver. 
Zahedi et al. demonstrated the induction of  the IL- 
Ira gene during inflammatory and acute phase reac- 
tions in the mouse liver, suggesting a possible involve- 
ment in the control of inflammatory mediators [10]. 
Many proteins involved in immune and inflammatory 
responses are expressed and/or controlled in the liver by 
an IL-1 stimulus mainly derived from macrophages. 
This includes several complement proteins such as C3, 
C5 and Factor B as well as acute phase proteins such 
as serum amyloid P component [11] and serum amyloid 
A [12]. It is necessary to control this IL-1 transcriptional 
activation in the liver in order to prevent an overexpres- 
sion which could lead to pathophysiological effects. 
Therefore, the existence of  a natural IL-lra expressed 
in the liver is essential and the knowledge of the eDNA 
sequence of this transcript opens the possibility to inves- 
tigate the specific control and the interaction with other 
proteins in the liver environment. The expression of the 
secreted form of the lL-lra in liver is consistent with the 
fact that this protein competes in the extraeellular envi- 
ronment with [L-I for the binding to IL-I receptors. 
Acknowledgements :  We would like to thank Dr K.B.M. Reid for 
helpful discussion and critisism of the manuscript. "rhi~ work is a 
contribution from the Oxford Centre of Molecular Science (OCMS) 
which is supported by SERC and the MRC. A.S. was supported by 
an OCMS post-doctoral fellowship. We also thank the Yamanouchi 
Research Institute UK for support. 
REFERENCES 
[1] Arend, W,P., Joslin, F.G. and Massoni, R.J. (1985) J. lmmunol. 
134, 3868-3875. 
[2] Dinarello, C.A. and Thompson, R.C, (1991) I mmnnol. Today i 2, 
404--410. 
[3] Eisenber 8,S.P,, Evans, g,J., Arend, W.P., Verderber, E., Brewer, 
M,T,, Haanum, C.H. and Thompson, R.C. (1990) Nature 343, 
341-346. 
[4] Steinkasserer, A., Koelble, K. and $im, R.B. (1991) Nueleie Acids 
Res. 19, 5095. 
[5] Steinkasserer, A., Spurt, N.K,  Cox, S., Jeggo, P. and S i re ,  R.B. 
(1992) Genomics 13, 654-657, 
[6] Lennard, A,, German, P., Carrier, M., Scotney, H., Sheer, D. and 
Solad, R. (1992) Cylokine 4, 83-89. 
[7] Haskill, S., Martin, G., Van Le, L., Morris, J ,  Peace, A., Bigler. 
C.F., Jafli~, G.J., Hammerberg, C., Spora, S.A., Forte, S., Arend, 
W.P. and Ralph, P. (1991) Prec. Natl, Aead. Sei. USA 88 ,  3681- 
3685. 
[8] Maniatis, T,, Frit~clt, E.F. and Sarnbrook, J. (1982) A Labora- 
tory Manual, Cold Spring Harbor Press, Cold Spring Harbor. 
61 
Volume 310, number 1 FEBS LETTERS September 1992 
[9] Estaller, C., Sehwaeble, W., Dierieh, M.P. and Weiss, E.H. (1991) 
Ear. J. Immunol. 21,799-802. 
[10] Zahedl, K., $eldin, M.F., Rim, M., Ezekowitz, A.B. and White- 
head, A.S. (1991) J. h'araun~l. 146, 4228-4233. 
[11] Zahdi, K. and Whitehead, A.S. (1989) J. lmmunol. 143, 2880- 
2886. 
[12] St¢inkassor~r, A.  Weiss, E.H., Schwaebl¢, W. and Link¢, R,P. 
(1990) Biochem. J. 268, 187-193. 
62 


Human interleukin-1 receptor antagonist is expressed in liver 

Open Access LMU

Volume 310, number 1, 60-62 FEBS 11476 © 1992 Federation of European Biochemicai Societies 00145793/92/$5.00 September 1992 Human interleukin-1 receptor antagonist is expressed in liver Alexander Steinkasserer~'~ Cornel ia Estaller b, El isabeth H. Weiss b and Robert  B. Sim:' "MRC bnmunochemistr), Unit, Department of Biochemistr.v, University of O.xford, South Parks Road, O.xford, OXI 3QU, UK and t'1nstitute of bnmtmology, University of Munich, Goethestr. 31, 8000 M~nich.2. German t,Received 14 July 1992 Using PCR and Northern blot analysis, an IL-I receptor antagonist pecific transcript was amplified from HepG2- and liver mRNA. eDNA clones codln8 for IL- 1 receptor antagonist were isolated from a liver eDNA library and sequence omparison revealed complete identity with the secreted, monocytie form of IL-1 receptor antagonist. IL-I receptor antagonist; Liver: eDNA transcript 1. INTRODUCTION IL-I receptor antagonist (IL-lra) is a protein which inhibits IL-1 (IL-I~ and IL-lfl) activity, by binding competitively to IL-I receptors [1]. This naturally oc- curring inhibitor has been shown to reduce the severity of sepsis, colitis, arthritis and diabetes in animals and it therefore plays an important role in the control of IL-I mediated inflammatory and autoimmune diseases [2]. IL-lra is structurally related to IL-1 and shows 18% and 26% identity in amino acid sequence to IL-I~ and li.-lfl, respectively. The eDNA sequence of IL-lra derived from mon- ocytes has been previously determined [3] and using an intronic polymorphism [4] the localization of the gone on chromosome 2 has been established by linkage anal- ysis [5], as well as by in situ hybridisation [6]. There are two types of IL-lra, one secreted, soluble form expressed in monocytes and netttrophils and an intracellular form expressed in epithelial cells. This in- tracellular IL-lra, which lacks a leader peptide, is de- rived from the same gene through the use of an alterna- tive transcriptional start site and alternative splicing [7]. In this study we report the expression of the secreted form of the lL-lra in liver. 2. MATERIALS AND METHODS 2.1. eDNA ~ynthesis and PCR amplification Total RNA was isolated from the human cell lines HepG2, Chang, U937, Daudi, HL60, Molt4, Raji and T47D as well as from liver, temporal cortex and lung tissue using guanidinium thiocyanate and a CsCI gradient [8]. 10/zg RNA was used for the first strand eDNA synthesis (eDNA synthesis kit; Amersham, Bucks, UK) and 1/10tb of this reaction mixture was used for PCR amplification. Th~ two PCR Correspondence address: A. Steinkasserer, MRC Immunoehemistry Unit, Dept. Biochemistry, South Parks Road, Oxford, OXI 3QU, UK. Fax: (44) (865) 275729 primer sets described by Haskill et al. [7] were used !n order to distin- guish between RNA transcripts for he secreted and the intracellular forms of t L-Ira. PCR products were analysed after 30 cycles (94°C, 1 min; 60"C, 30 s, 72°C, 2 rain) on a 0.7% (w/v) agarose gel. 2.2. Northern blot ana6,~'is A 10/.tg portion of total RNA was electrophoresed in 1.2% (w/v) agarose gel and the RNA transferred to a Hybond-N membrane (Amersham) and hybridised at 65°C with a radiolabelled IL-lra probe dei'ived fi'om HepG2 mRNA b2¢ PCR. The blots were washed at high stringency [8]. 2.3. Isolation of lL-lra clones and eDNA sequencing A human liver cDNA library constructed in the eukaryotic expres- sion vector CDM8 [9] was screened with an IL-lra probe derived from HepG2 mRNA by PCR. The length of the positive cDNA clones was estimz ..d by a Pstl.Hindlll restriction digest. The sequence was deter- mined from both strands using the double-strand sequencing kit from Pharmacia (Milton Keyne~, UK), 3. RESULTS AND DISCUSSION Using specific PCR primers it was possible to amplify an IL- Ira transcript from the hepatoma cell line HepG2 and from total liver RNA. In liver on!y the secreted form of the antagonist was present (Table I) and no amplification product was obtained for the intracellular form. Interestingly no amplification product was ob- tained from the Chang liver cell line which is derived from nonmalignant human liver tissue, indicating a loss of the IL-Ira message in this cell line or need for an external stimulus. As a positive control the monocytic cell line U937 was used. The other cell lines (Molt4, HL60, Daudi, T47D and Raji) and tissues (temporal cortex and lung) were negative for the secreted (Table I) as well as for the intracellular IL-lra (data not shown). To confirm the PCR analysis, Northern blots were performed with RNA extracted from liver tissue and from the cell lines HepG2, Chang and Raji. As shown 60 P, blished by Elsevier Science Publishers B. V. Volume 310, number 1 FEBS LETTERS September 1992 I(, 0 I~i H C R ,~.~'-' ~ . i " . : " . : . " : " ' , : ,  :~' ."  ~- :~"  , . ,...-.:~..:, -:-: :~  : . : . , " . . . , . ; . . ' :  .~ ; . . ,  . . . . . .  . : . . - r . , ,2  :: . . . - : : : . . . . ' .  ,..,. :.. i:~i::i:'?: : " '  : ~:.~;~'-: ::/i~::i~' ;,.i:~;¢:.. ! . . . .  : ":.-.:; ::!~ ? ,::6: / ~ ~, : ' . c  - " ~. '~  " • " " ,  ~ .  ~Z'-.~.' , : : ? i~ ; "  '-~ :, ~ ~'~": :.~:.... ~ :":t'~!':  ~ : : :~ , '  • .:.~ : ; , : :  : .  ~':,~0'. . .  ' . - . .~- ,  : - : - . ' : ! . . .~  " . ,  .~::::'- -~'~ .. :,~-~ . :~  . . . .  . . .  ' ~ . . . . . . .  ; - . . . . . . . . . . .  ,.: • '. ;i~77~.: 7~;? .~. . .  : - ..~-. • ¢!~ ; 5~;.i f.~ . : ..r " : : . . , : :~ . :  ~ ~::: " , . ~  • . . . .  • . .. . • . . . . . : ; . , . .  , y  , . ' . . ' ,~ ; / ,  , ' . i  ~ '  " :  . . . . . . .  . . . .  : -~  ,i ~: : "?  ~"  " . ' "  ; " - ' . f .?  • .! i .(:. . : " ' .~ :~ " , ? i  r~ -i,~. ~:,:';,..,, ~ . : : ,~ . . . , ,~  .~.,~ ' ; i : : , ; ; ' . - " ;  "~: -  ¢ : " :~. .  " - "  / . ' . ' 7£ : -~: : ' " ' ? . " .  I L - I  RA- -  Fig. 1. Northern blot analysis of total RNA isolated from liver tissue (lanes 1-3; individuals: K, O, N,) and the cell lines HepG2 (H), Chang (C) and gaji (R) probed with an IL-lra eDNA probe. The additional fainter bands are probably due to background-hybridization or ot totally spliced mRNA. in Fig. 1 all three liver RNAs (individuals: K, O, N) contained the IL-lra transcript (lanes 1-3), as did the HepG2 cell line (lane 4). No IL-I ra transcript was de- tected in the cell lines Chang and Raji (lanes 5 and 6), in agreement with the findings obtained by PCR analy- sis. To establish the sequence of this liver IL-lra tran- script a liver cDNA library subeloned into the CDM8 expression vector was screened with an IL-lra probe. Three positive clones were isolated and a 1.65 kb insert from clone plLlra-L was sequenced. This clone con- tains a short Y-untranslated region (8 bp) followed by Table I PCR analysis of cell line and tissue eDNA transcripts sec. IL-I ra HepG2 Chang Liver U937 Daudi HL60 Molt4 gaji T47D Cortex Lung (hepatoma) + (hepatocyte) (tissue) + (monoeytie) + (B.lymphoblastoid) (net,trphilic) (T-lymphoblastoid) (B-lymphobla~toid) (breast carcinoma) (tissue) (tissue) the leader sequence and the coding region for the ma- ture secreted protein. The T-untranslated region is 1108 bp long. Sequence comparison revealed that this se- quen~ is identical to the eDNA corresponding to the secreted form of the 1L-lra from monocytes [3]. Since the IL-ra transcript was detected (by PCR and Northern blot) in liver tissue as well as in the hepatoma cell line HepG2 the transcript in the tissue is likely to be derived from hepatocytes and therefore represents atrue IL-lra transcript in human liver. Zahedi et al. demonstrated the induction of  the IL- Ira gene during inflammatory and acute phase reac- tions in the mouse liver, suggesting a possible involve- ment in the control of inflammatory mediators [10]. Many proteins involved in immune and inflammatory responses are expressed and/or controlled in the liver by an IL-1 stimulus mainly derived from macrophages. This includes several complement proteins such as C3, C5 and Factor B as well as acute phase proteins such as serum amyloid P component [11] and serum amyloid A [12]. It is necessary to control this IL-1 transcriptional activation in the liver in order to prevent an overexpres- sion which could lead to pathophysiological effects. Therefore, the existence of  a natural IL-lra expressed in the liver is essential and the knowledge of the eDNA sequence of this transcript opens the possibility to inves- tigate the specific control and the interaction with other proteins in the liver environment. The expression of the secreted form of the lL-lra in liver is consistent with the fact that this protein competes in the extraeellular envi- ronment with [L-I for the binding to IL-I receptors. Acknowledgements :  We would like to thank Dr K.B.M. Reid for helpful discussion and critisism of the manuscript. "rhi~ work is a contribution from the Oxford Centre of Molecular Science (OCMS) which is supported by SERC and the MRC. A.S. was supported by an OCMS post-doctoral fellowship. We also thank the Yamanouchi Research Institute UK for support. REFERENCES [1] Arend, W,P., Joslin, F.G. and Massoni, R.J. (1985) J. lmmunol. 134, 3868-3875. [2] Dinarello, C.A. and Thompson, R.C, (1991) I mmnnol. Today i 2, 404--410. [3] Eisenber 8,S.P,, Evans, g,J., Arend, W.P., Verderber, E., Brewer, M,T,, Haanum, C.H. and Thompson, R.C. (1990) Nature 343, 341-346. [4] Steinkasserer, A., Koelble, K. and $im, R.B. (1991) Nueleie Acids Res. 19, 5095. [5] Steinkasserer, A., Spurt, N.K,  Cox, S., Jeggo, P. and S i re ,  R.B. (1992) Genomics 13, 654-657, [6] Lennard, A,, German, P., Carrier, M., Scotney, H., Sheer, D. and Solad, R. (1992) Cylokine 4, 83-89. [7] Haskill, S., Martin, G., Van Le, L., Morris, J ,  Peace, A., Bigler. C.F., Jafli~, G.J., Hammerberg, C., Spora, S.A., Forte, S., Arend, W.P. and Ralph, P. (1991) Prec. Natl, Aead. Sei. USA 88 ,  3681- 3685. [8] Maniatis, T,, Frit~clt, E.F. and Sarnbrook, J. (1982) A Labora- tory Manual, Cold Spring Harbor Press, Cold Spring Harbor. 61 Volume 310, number 1 FEBS LETTERS September 1992 [9] Estaller, C., Sehwaeble, W., Dierieh, M.P. and Weiss, E.H. (1991) Ear. J. Immunol. 21,799-802. [10] Zahedl, K., $eldin, M.F., Rim, M., Ezekowitz, A.B. and White- head, A.S. (1991) J. h'araun~l. 146, 4228-4233. [11] Zahdi, K. and Whitehead, A.S. (1989) J. lmmunol. 143, 2880- 2886. [12] St¢inkassor~r, A.  Weiss, E.H., Schwaebl¢, W. and Link¢, R,P. (1990) Biochem. J. 268, 187-193. 62 

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Human interleukin-1 receptor antagonist is expressed in liver

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