Corticosteroid-independent inhibition of tumor necrosis factor
production by the neuropeptide urocortin
Davide
Agnello1,
Riccardo
Bertini2,
Silvano
Sacco1,
Cristina
Meazza1,
Pia
Villa1,3, and
Pietro
Ghezzi1
1 "Mario Negri" Institute
for Pharmacological Research, 20157 Milan;
2 Department of Pharmacology,
Dompé SpA Research Center, 67100 L'Aquila; and
3 Consiglio Nazionale delle
Ricerche-Cellular and Molecular Pharmacology Center, 20129 Milan,
Italy
 |
ABSTRACT |
Urocortin (UCN) is a
neuropeptide homologous with corticotropin-releasing factor (CRF),
which has anti-inflammatory activities not all mediated by
corticosteroids. In mice, UCN (1 µg/mouse sc) significantly
reduced lipopolysaccharide (LPS)-induced serum tumor necrosis factor
(TNF) and interleukin (IL)-1
levels in vivo but did not affect serum
IL-6. These effects were paralleled by a rise in corticosterone (CS)
levels. Blockade of the CS increase by cyanoketone did not prevent TNF
inhibition by UCN, suggesting the neuropeptide has anti-inflammatory
mechanisms independent of the hypothalamus-pituitary-adrenal axis. In
fact UCN had a direct inhibitory effect on LPS-induced TNF in rat
Kupffer cells at concentrations between
10
10 and
10
16 M, and this effect was
related to increased cAMP levels. However, the in vivo inhibition of
LPS-induced IL-1
by UCN was reversed by cyanoketone, indicating that
the increase of endogenous glucocorticoids might be more important in
IL-1
inhibition than in TNF inhibition by UCN.
inflammation; cytokines; corticotropin-releasing factor; lipopolysaccharide; hypothalamus-pituitary-adrenal axis; interleukin-1; interleukin-6; Kupffer cells
 |
INTRODUCTION |
UROCORTIN (UCN), a recently described neuropeptide
detected in rat brain and 45% homologous with corticotropin-releasing
factor (CRF) (28), is an endogenous ligand for the CRF receptor. The main physiological role of CRF is activation of the
hypothalamus-pituitary-adrenal axis (HPAA), where it stimulates ACTH
production by the pituitary and, thus, ultimately increases the release
of corticosteroids. CRF reportedly acts by binding to two different
receptors. CRF receptor type 1 is expressed mainly in the brain and the
pituitary (18), whereas CRF receptor type 2 is expressed in brain and peripheral tissues but not in the pituitary (15). The distribution of
UCN is not the same as CRF and correlates with the distribution of the
CRF receptor type 2 but not type 1 (28); UCN also has higher affinity
for receptor type 2 than CRF itself (28).
Because corticosteroids are potent anti-inflammatory agents, UCN and
CRF can be viewed as anti-inflammatory mediators. In fact,
immunosuppressive or anti-inflammatory effects of exogenously administered CRF have been described (6, 13, 17, 31), and CRF
transgenic mice have an immunosuppressive phenotype that is reversed by
adrenalectomy (3). The mechanism of anti-inflammatory action of CRF is
unclear, and it may have anti-inflammatory activities independent of
endogenous corticosteroids (12, 17).
UCN is also reported to have anti-inflammatory activity (27) and to
inhibit experimental autoimmune encephalomyelitis (17) by a
glucocorticoid-independent mechanism. Its anti-inflammatory activity
may be mediated by CRF receptor type 2 (27).
Various cytokines are considered important pathogenic mediators of
inflammation. Tumor necrosis factor (TNF) is particularly important, as
demonstrated by the protective effects of anti-TNF antibodies or
inhibitors of TNF synthesis in animal models of arthritis (32) and
experimental autoimmune encephalomyelitis (22) and by the clinical
efficacy of anti-TNF antibodies in rheumatoid arthritis (8).
Corticosteroids are potent inhibitors of TNF synthesis, so activation
of the HPAA amounts to a negative-feedback mechanism that limits TNF
synthesis (5, 11). In fact, activation of the HPAA by cytokines or
stress inhibits TNF production (10), whereas its blockade by
adrenalectomy or glucocorticoid antagonists or inhibitors upregulates
TNF production and worsens TNF-mediated diseases (2, 11).
To better define the effects of these neuropeptides in inflammation, we
studied the effect of UCN on TNF, using a model where lipopolysaccharide (LPS) is administered to mice to induce circulating TNF. We also investigated the levels of two other inflammatory cytokines, interleukin (IL)-1
and IL-6.
Because these experiments showed that UCN had an inhibitory effect on
TNF and IL-1
levels, we investigated whether this was a direct or
HPAA-mediated effect, using cyanoketone to prevent the rise of blood
corticosterone (CS) (10). Finally, we investigated whether UCN directly
affected TNF levels in mouse blood or rat Kupffer cells in vitro.
 |
MATERIALS AND METHODS |
Materials. Synthetic rat UCN was
synthesized and kindly provided by Dr. Nicholas Ling of Neurocrine
Biosciences (San Diego, CA). Synthetic rat-human CRF and LPS
(Escherichia coli 055:B5) were from
Sigma (St Louis, MO). Cyanoketone was a kind gift from Sanofi Research
Division (Malvern, PA).
Animals and treatments. Male Crl:CD-1
(ICR) BR mice (25 g body wt) were used for in vivo studies.
Male Crl:CD (SD) BR rats (200-250 g body wt) were used for
preparation of Kupffer cells. Procedures involving animals and their
care were conducted in conformity with the institutional guidelines
that are in compliance with national (D.L. no. 116, G.U., suppl. 40, 18 Febbraio 1992, Circolare no. 8, G.U., 14 Luglio 1994) and international
(EEC Council Directive 86/609, OJL 358, 1, December 12, 1987; Guide for
the Care and Use of Laboratory Animals, US National Research Council,
1996) laws and policies.
UCN was reconstituted by adding 0.1 ml of 1 N acetic acid to 1 mg of
peptide. CRF was dissolved in sterile, pyrogen-free water to a
concentration of 1 mg/ml. Both peptides were then diluted in
phosphate-buffered saline (PBS) and injected at the dose of 1 µg/mouse sc. Control mice received PBS alone. One hour later, LPS was
injected at the dose of 100 ng/mouse iv. Serum TNF and IL-6 were
determined at 1.5 h, and serum IL-1
was determined at 3 h after LPS
injection. These schedules were chosen on the basis of the kinetics of
cytokine induction after LPS (4, 24) and of preliminary experiments. CS
was determined at the times of LPS injection (1 h after UCN and CRF)
and cytokine determination (1.5 and 3 h after LPS) on different groups
of animals. Control mice received saline alone. Blood was obtained from
the retroorbital plexus, and serum was prepared and stored frozen until
it was used for cytokine and CS assays.
In some experiments, cyanoketone was given twice, 24 h and 1.5 h before
the experiment, at the dose of 100 mg/kg ip in corn oil (10). Control
mice received the same amount of corn oil.
Whole blood cultures. For whole blood
cultures, heparinized (14 U/ml; Liquemin, Roche, Milan, Italy), freshly
obtained whole blood was plated in 96-well tissue culture plates (100 µl/well) and incubated for 4 h at 37°C in 5%
CO2, with 1 µg/ml LPS
and with and without different concentrations of UCN (added 1 h before LPS). At the end of the incubation, blood was diluted 1:1 (vol/vol) with cold RPMI 1640 medium and centrifuged, and the supernatant was
collected for TNF determination.
Kupffer cell cultures and cAMP
determination. Kupffer cells were prepared according to
the method of Smedsrod and Pertoft (25) after perfusion and digestion
of rat liver. Cells were seeded at 0.2 × 106/well for TNF experiments and
at 4 × 106/well for cAMP and
were used after 48 h of culture in Williams' medium E at 37°C in a
5% CO2-humidified atmosphere.
For TNF determination, the cells were incubated at 37°C for 30 min
with either control medium or UCN at different concentrations and then
stimulated with 0.1 µg/ml of LPS. After 4 h, the supernatants were
collected for TNF assay.
For cAMP determination, the cells were incubated at 37°C for 30 min
with 1 mM 3-isobutyl-1-methylxanthine (Sigma) and for another 30 min
with either control medium or UCN at different concentrations. The
cells were then placed on ice, and the medium was removed. Cultures
were washed twice with cold PBS and scraped into 10 ml of cold 65%
(vol/vol) ethanol. After centrifugation the supernatants were dried
under a stream of nitrogen at 60°C, and the dried extracts were
dissolved in 1 ml of assay buffer for cAMP determination with cAMP
ELISA from Amersham International (Little Chalfont, UK), according to
the instructions of the manufacturer.
Cytokine determination. TNF was
measured by cytotoxicity on L929 cells as previously described (1),
using recombinant TNF-
as a standard (specific activity 0.6 × 107 U/mg; a kind gift from BASF,
Ludwigshafen, Germany). The sensitivity of the assay was 0.1 U/ml. IL-6
was measured as hybridoma growth factor with 7TD1 cells (a kind gift
from Dr. van Snick) as previously described (23). IL-6 activity is
expressed as costimulatory units per milliliter, using recombinant IL-6
as a standard. The sensitivity of the assay was 50 U/ml.
IL-1
was measured with an ELISA kit (Amersham International). The
sensitivity of the assay was 3 pg/ml.
CS determination. CS was measured by a
competitive-binding RIA, with an antiserum for corticosterone RIA
obtained from Sigma (C-8784), as previously described (10).
[3H]corticosterone was
purchased from Amersham International.
 |
RESULTS |
As shown in Fig.
1A, UCN
or CRF at the dose of 1 µg/mouse, 1 h before LPS,
significantly reduced serum TNF levels (85 and 75% inhibition,
respectively). UCN also lowered serum levels of IL-1
by 65%,
whereas CRF had no effect (Fig. 1B).
IL-6 was unaffected by either UCN or CRF (Fig.
1C). No TNF, IL-1
, or IL-6 was
detected in serum of control mice (data not shown).

View larger version (22K):
[in this window]
[in a new window]

View larger version (31K):
[in this window]
[in a new window]

View larger version (29K):
[in this window]
[in a new window]
|
Fig. 1.
Effect of urocortin (UCN) and corticotropin-releasing factor (CRF) on
tumor necrosis factor (TNF; A),
interleukin (IL)-1 (B), and IL-6
(C) levels. Mice were treated with
UCN or CRF (1 µg/mouse sc). Control mice received PBS alone.
Lipopolysaccharide (LPS; 100 ng/mouse iv) was given 1 h later. Serum
TNF and IL-6 were measured 1.5 h after LPS; serum
IL-1 was measured 3 h after LPS. Data are means ± SD (5 mice/group). * P < 0.05, ** P < 0.01 vs. PBS alone by
Duncan's test.
|
|
We also evaluated the effect of UCN or CRF on serum CS levels, which
were increased 1 h after UCN or CRF (Fig.
2), in agreement with previous results
(21). LPS induced CS at higher levels than UCN or CRF alone, and
pretreatment of mice with these neuropeptides did not further increase
the response to LPS (Fig. 2).

View larger version (33K):
[in this window]
[in a new window]
|
Fig. 2.
Effect of UCN and CRF on corticosterone (CS) levels. Mice were treated
with UCN or CRF (1 µg/mouse sc). Control mice received PBS alone.
Serum CS was measured 1 h after UCN or CRF alone and 1.5 h after LPS
with (+) or without ( ) UCN or CRF. Data are means ± SD (5 mice/group).
** P < 0.01 vs. PBS alone by
Duncan's test. § P < 0.01 vs. PBS, UCN, or CRF alone (without LPS) by Duncan's test.
|
|
To investigate whether TNF and IL-1
inhibition by UCN was fully
mediated by the increase of glucocorticoids, we inhibited UCN- and
LPS-induced CS synthesis by pretreating mice with cyanoketone (10)
(Figs. 3B
and 4B).
As shown in Figs. 3A and
4A, UCN significantly reduced TNF
levels but did not affect IL-1
production in cyanoketone-pretreated mice.

View larger version (21K):
[in this window]
[in a new window]

View larger version (22K):
[in this window]
[in a new window]
|
Fig. 3.
Effect of cyanoketone on UCN inhibition of TNF
(A) and CS
(B) levels. Mice were treated with
UCN (1 µg/mouse sc). Control mice received PBS alone. Cyanoketone was
given intraperitoneally at the dose of 100 mg/kg, 24 h and 1.5 h before
UCN (or PBS). LPS (100 ng/mouse iv) was given 1 h after UCN or PBS.
Serum TNF and CS were measured 1.5 h after LPS. Data are means ± SD
(5 mice/group). * P < 0.05, ** P < 0.01 vs. PBS control by
Duncan's test.
|
|

View larger version (32K):
[in this window]
[in a new window]

View larger version (17K):
[in this window]
[in a new window]
|
Fig. 4.
Effect of cyanoketone on UCN inhibition of IL-1
(A) and CS
(B) levels. Mice were treated with
UCN (1 µg/mouse sc). Control mice received PBS alone. Cyanoketone was
given intraperitoneally at the dose of 100 mg/kg, 24 h and 1.5 h before
UCN (or PBS). LPS (100 ng/mouse iv) was given 1 h after UCN or PBS.
Serum IL-1 and CS were measured 3 h after LPS. Data are means ± SD (5 mice/group). * P < 0.05, ** P < 0.01 vs.
PBS control by Duncan's test.
|
|
To study the direct effects of UCN on TNF-producing cells, we performed
a series of in vitro experiments on whole mouse blood and rat Kupffer
cell cultures. On whole mouse blood at UCN concentrations between
10
7 and
10
14 M, we never observed
more than 30% inhibition, which was not statistically significant
(data not shown). However, UCN significantly inhibited TNF production
by rat Kupffer cells with an inverted, bell-shaped dose-response curve.
The effective concentrations ranged between
10
10 and
10
16 M (Fig.
5).

View larger version (17K):
[in this window]
[in a new window]
|
Fig. 5.
Effect of UCN on TNF production by rat Kupffer cells. Kupffer cells
were incubated for 30 min in the presence of UCN at different
concentrations and then stimulated with 0.1 µg/ml of LPS. TNF levels
were determined in medium after 4 h. Data are means ± SD from
triplicate wells. * P < 0.05, ** P < 0.01 vs. LPS alone.
|
|
To further investigate the CS-independent mechanism for UCN inhibition
of TNF production, we also measured cAMP levels in rat Kupffer cells
after exposure to different UCN concentrations. The neuropeptide
significantly raised cAMP levels at the concentrations that inhibited
TNF production (Table 1).
 |
DISCUSSION |
The present study showed that UCN, like CRF, inhibits LPS-induced TNF
in vivo. This effect seems to be largely independent of endogenous CS.
In fact, UCN also inhibited TNF levels in cyanoketone-pretreated mice,
although to a slightly lesser extent than in intact mice (in three
independent experiments, UCN inhibited TNF production by 85, 83 and
76%; in two experiments with cyanoketone-pretreated mice, inhibition
amounted to 70 and 57%).
The corticosteroid-independent immunosuppressive action of UCN is in
agreement with a report that this neuropeptide can protect adrenalectomized animals from experimental autoimmune encephalomyelitis (17). A direct effect on TNF-producing cells might be the cause of UCN
inhibition of TNF production, although this does not rule out the
possibility that other mediators produced by the HPAA reported to
inhibit TNF production, such as the peptide melanocyte-stimulating hormone (14), might be important too.
We focused our attention on the first hypothesis and tried to
demonstrate a direct effect on TNF production in vitro in mouse blood
and rat Kupffer cells. In our experimental conditions, we observed no
real effect of UCN in whole blood. However, UCN significantly inhibited
TNF production in rat Kupffer cells, which constitute the main
macrophage population of the body. This observation is in agreement
with the reported presence of CRF receptors on macrophages (7).
We also investigated the possibility that cAMP was involved in the
mechanism of TNF inhibition by UCN. UCN, like CRF (30), has been
reported to raise cAMP levels (28), and cAMP is a potent inhibitor of
TNF production (20, 26). There was an increase in the amount of cAMP in
Kupffer cells after pretreatment with UCN at concentrations that lower
TNF levels. Further studies are needed to see whether this increase in
cAMP is the only mechanism responsible for the inhibition of TNF production.
We have also shown that UCN inhibits IL-1
production, but this
effect is reversed by cyanoketone. The increase of endogenous glucocorticoids might therefore be more important in IL-1
inhibition by UCN than in TNF inhibition, and the increase in cAMP levels might be
less important than in the case of TNF inhibition. In fact, although
there is no doubt about the effects of cAMP on TNF production,
conflicting reports have been published regarding the inhibition of
IL-1 production by agents that increase cAMP levels. Some authors
reported that IL-1 production was only slightly or not at all inhibited
in human monocytes or mouse macrophages pretreated with
phosphodiesterase inhibitors (9, 16, 19), whereas others reported that
rolipram reduced IL-1
secretion from human monocytes but increased
IL-1
mRNA (29). In a preliminary experiment, we did not see any
inhibition of IL-1 secretion from Kupffer cells pretreated with UCN at
different concentrations (data not shown).
Finally, we report that UCN does not affect IL-6. This is consistent
with previous data showing that IL-6 levels in vivo are not inhibited
and may be even increased by cAMP-elevating agents (33, 34). IL-6
production in vivo is also resistant to the inhibitory effect of
glucocorticoids, whereas TNF production is extremely sensitive even to
low doses of glucocorticoids (24).
In conclusion, we have shown that UCN is a potent inhibitor of TNF
production in vivo and in vitro and also inhibits production of IL-1
in vivo, an observation that supports the concept of an
anti-inflammatory activity of this neuropeptide, through
corticosteroid-independent and -dependent mechanisms.
 |
ACKNOWLEDGEMENTS |
D. Agnello is a recipient of a fellowship from Fondazione A. and A. Valenti.
 |
FOOTNOTES |
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests: P. Ghezzi, "Mario Negri" Institute
for Pharmacological Research, via Eritrea 62, 20157 Milan, Italy.
Received 17 February 1998; accepted in final form 28 July 1998.
 |
REFERENCES |
1.
Aggarwal, B. B.,
W. J. Khor,
P. E. Hass,
B. Moffat,
S. A. Spencer,
J. Henzel,
S. Bringman,
G. E. Nedwin,
D. V. Goeddel,
and
R. N. Harkins.
Human tumor necrosis factor. Production, purification and characterization.
J. Biol. Chem.
260:
2345-2354,
1985[Abstract].
2.
Bertini, R.,
M. Bianchi,
and
P. Ghezzi.
Adrenalectomy sensitizes mice to the lethal effects of interleukin 1 and tumor necrosis factor.
J. Exp. Med.
167:
1708-1712,
1988[Abstract].
3.
Boehme, S. A.,
A. Gaur,
P. D. Crowe,
X. J. Liu,
S. Tamraz,
T. Wong,
A. Pahuja,
N. Ling,
W. Vale,
E. B. De Souza,
and
P. J. Conlon.
Immunosuppressive phenotype of corticotropin-releasing factor transgenic mice is reversed by adrenalectomy.
Cell. Immunol.
176:
103-112,
1997[Medline].
4.
Chensue, S. W.,
P. D. Terebuh,
D. G. Remick,
W. E. Scales,
and
S. L. Kunkel.
In vivo biologic and immunohistochemical analysis of interleukin-1 alpha, beta and tumor necrosis factor during experimental endotoxemia.
Am. J. Pathol.
138:
395-402,
1991[Abstract].
5.
Chrousos, G. P.
The hypothalamic-pituitary-adrenal axis and immune-mediated inflammation.
N. Engl. J. Med.
332:
1351-1362,
1995[Free Full Text].
6.
Correa, S. G.,
C. M. Riera,
J. Spiess,
and
I. D. Bianco.
Modulation of the inflammatory response by corticotropin-releasing factor.
Eur. J. Pharmacol.
319:
85-90,
1997[Medline].
7.
Dave, J. R.,
L. E. Eiden,
and
R. L. Eskay.
Corticotropin-releasing factor binding to peripheral tissue and activation of the adenylate cyclase-adenosine 3',5'-monophosphate system.
Endocrinology
116:
2152-2159,
1985[Abstract].
8.
Elliott, M. J.,
R. N. Maini,
M. Feldmann,
J. R. Kalden,
C. Antoni,
J. S. Smolen,
B. Leeb,
F. C. Breedveld,
J. D. Macfarlane,
H. Bijl,
and
J. N. Woody.
Randomised double-blind comparison of chimeric monoclonal antibody to tumour necrosis factor
(cA2) versus placebo in rheumatoid arthritis.
Lancet
344:
1105-1110,
1994[Medline].
9.
Endres, S.,
H.-J. Fulle,
B. Sinha,
D. Stoll,
C. A. Dinarello,
R. Gerzer,
and
P. C. Weber.
Cyclic nucleotides differentially regulate the synthesis of tumour necrosis factor-
and interleukin-1
by human mononuclear cells.
Immunology
72:
56-60,
1991[Medline].
10.
Fantuzzi, G.,
E. Di Santo,
S. Sacco,
F. Benigni,
and
P. Ghezzi.
Role of the hypothalamus-pituitary-adrenal axis in the regulation of tumor necrosis factor production in mice: effect of stress and inhibition of endogenous glucocorticoids.
J. Immunol.
155:
3552-3555,
1995[Abstract].
11.
Fantuzzi, G.,
and
P. Ghezzi.
Glucocorticoids as cytokine inhibitors: role in neuroendocrine control and therapy of inflammatory diseases.
Mediat. Inflamm.
2:
263-270,
1993.
12.
Kelley, D. M.,
A. Lichtenstein,
J. Wang,
A. N. Taylor,
and
S. M. Dubinett.
Corticotropin-releasing factor reduces lipopolysaccharide-induced pulmonary vascular leak.
Immunopharmacol. Immunotoxicol.
16:
139-148,
1994[Medline].
13.
Labeur, M. S.,
E. Artz,
G. J. Wiegers,
F. Holsboer,
and
J. M. Reul.
Long-term intracerebroventricular corticotropin-releasing hormone administration induces distinct changes in rat splenocyte activation and cytokine expression.
Endocrinology
136:
2678-2688,
1995[Abstract].
14.
Lipton, J. M.,
and
A. Catania.
Anti-inflammatory actions of the neuroimmunomodulator
-MSH.
Immunol. Today
18:
140-145,
1997[Medline].
15.
Lovenberg, T.,
D. Chalmers,
C. Liu,
and
E. De Souza.
CRF2
and CRF2
receptor mRNAs are differentially distributed between the rat central nervous system and peripheral tissues.
Endocrinology
136:
4139-4142,
1995[Abstract].
16.
Molnar-Kimber, K. L.,
L. Yonno,
R. J. Heaslip,
and
B. M. Weichman.
Differential regulation of TNF-
and IL-1
production from endotoxin stimulated human monocytes by phosphodiesterase inhibitors.
Mediat. Inflamm.
1:
411-417,
1992.
17.
Poliak, S.,
F. Mor,
P. Conlon,
T. Wong,
N. Ling,
J. Rivier,
W. Vale,
and
L. Steinman.
The neuropeptides corticotropin-releasing factor and urocortin suppress encephalomyelitis via effects on both the hypothalamic-pituitary-adrenal axis and the immune system.
J. Immunol.
158:
5751-5756,
1997[Abstract].
18.
Potter, E.,
S. Sutton,
C. Donaldson,
R. Chen,
M. Perrin,
K. Lewis,
P. Sawchenko,
and
W. Vale.
Distribution of corticotropin-releasing factor receptor mRNA expression in the rat brain and pituitary.
Proc. Natl. Acad. Sci. USA
91:
8777-8781,
1994[Abstract].
19.
Prabhakar, U.,
D. Lipshutz,
J. O. Bartus,
M. J. Slivjak,
E. F. Smith,
J. C. Lee,
and
K. M. Esser.
Characterization of cAMP-dependent inhibition of LPS-induced TNF alpha production by rolipram, a specific phosphodiesterase IV (PDE IV) inhibitor.
Int. J. Immunopharmacol.
16:
805-816,
1994[Medline].
20.
Renz, H.,
J.-H. Gong,
A. Schmidt,
M. Nain,
and
D. Gemsa.
Release of tumor necrosis factor-
from macrophages. Enhancement and suppression are dose-dependently regulated by prostaglandin E2 and cyclic nucleotides.
J. Immunol.
141:
2388-2393,
1988[Abstract/Free Full Text].
21.
Rivier, C.,
M. Brownstein,
J. Spiess,
J. Rivier,
and
W. Vale.
In vivo corticotropin-releasing factor-induced secretion of adrenocorticotropin,
-endorphin, and corticosterone.
Endocrinology
110:
272-278,
1982[Abstract].
22.
Ruddle, N. H.,
C. M. Bergman,
K. M. McGrath,
E. G. Lingenheld,
M. L. Grunnet,
and
S. J. Padula.
An antibody to lymphotoxin and tumor necrosis factor prevents transfer of experimental allergic encephalomyelitis.
J. Exp. Med.
172:
1193-1200,
1990[Abstract].
23.
Sironi, M.,
F. Breviario,
P. Proserpio,
A. Biondi,
A. Vecchi,
J. Van Damme,
E. Dejana,
and
A. Mantovani.
IL-1 stimulates IL-6 production in endothelial cells.
J. Immunol.
142:
549-553,
1989[Abstract/Free Full Text].
24.
Sironi, M.,
M. Gadina,
M. Kankova,
F. Riganti,
A. Mantovani,
M. Zandalasini,
and
P. Ghezzi.
Differential sensitivity of in vivo TNF and IL-6 production to modulation by anti-inflammatory drugs in mice.
Int. J. Immunopharmacol.
14:
1045-1050,
1992[Medline].
25.
Smedsrod, B.,
and
H. Pertoft.
Preparation of pure hepatocytes and reticuloendothelial cells in high yield from a single rat liver by means of Percoll centrifugation and selective adherence.
J. Leukoc. Biol.
38:
213-230,
1985[Abstract].
26.
Strieter, R. M.,
D. G. Remick,
P. A. Ward,
R. M. Spengler,
J. P. Lynch,
J. Larrick,
and
S. M. Kunkel.
Cellular and molecular regulation of tumor necrosis factor-alpha production by pentoxifylline.
Biochem. Biophys. Res. Commun.
155:
1230-1236,
1988[Medline].
27.
Turnbull, A. V.,
W. Vale,
and
C. Rivier.
Urocortin, a corticotropin-releasing factor-related mammalian peptide, inhibits edema due to thermal injury in rats.
Eur. J. Pharmacol.
303:
213-216,
1996[Medline].
28.
Vaughan, J.,
C. Donaldson,
J. Bittencourt,
M. H. Perrin,
K. Lewis,
S. Sutton,
R. Chan,
A. V. Turnbull,
D. Lovejoy,
C. Rivier,
J. Rivier,
P. E. Sawchenko,
and
W. Vale.
Urocortin, a mammalian neuropeptide related to fish urotensin I and to corticotropin-releasing factor.
Nature
378:
287-292,
1995[Medline].
29.
Verghese, M. W.,
R. T. McConnell,
A. B. Strickland,
R. C. Gooding,
S. A. Stimpson,
D. P. Yarnall,
D. J. Taylor,
and
P. J. Furdon.
Differential regulation of human monocyte-derived TNF alpha and IL-1 beta by type IV cAMP-phosphodiesterase (cAMP-PDE) inhibitors.
J. Pharmacol. Exp. Ther.
272:
1313-1320,
1995[Abstract].
30.
Webster, E. L.,
G. Battaglia,
and
E. B. De Souza.
Functional corticotropin-releasing factor (CRF) receptors in mouse spleens: evidence from adenylate cyclase studies.
Peptides
10:
395-401,
1989[Medline].
31.
Wei, E. T.,
S. Serda,
and
J. Q. Tian.
Protective actions of corticotropin-releasing factor on thermal injury to rat pawskin.
J. Pharmacol. Exp. Ther.
247:
1082-1085,
1988[Abstract].
32.
Williams, R. O.,
M. Feldmann,
and
R. M. Maini.
Anti-tumor necrosis factor ameliorates joint disease in murine collagen-induced arthritis.
Proc. Natl. Acad. Sci. USA
89:
9784-9788,
1992[Abstract].
33.
Zeni, F.,
P. Pain,
M. Vindimian,
J.-P. Gay,
P. Gery,
M. Bertrand,
Y. Page,
D. Page,
R. Vermesch,
and
J.-C. Bertrand.
Effects of pentoxifylline on circulating cytokine concentrations and hemodynamics in patients with septic shock: results from a double-blind, randomized, placebo-controlled study.
Crit. Care Med.
24:
207-214,
1996[Medline].
34.
Zhang, Y.,
J.-X. Lin,
and
J. Vilcek.
Synthesis of interleukin 6 (interferon-beta 2/B cell stimulatory factor 2) in human fibroblasts is triggered by an increase in intracellular cyclic AMP.
J. Biol. Chem.
263:
6177-6182,
1988[Abstract/Free Full Text].
Am J Physiol Endocrinol Metab 275(5):E757-E762
0002-9513/98 $5.00
Copyright © 1998 the American Physiological Society