ARTICLE
Auteur(s) :, Davide Agnello1, Patrizia
Mascagni3, Riccardo Bertini3, Pia
Villa1,2, Giorgio Senaldi4, Pietro Ghezzi1,*
1« Mario Negri » Institute for
Pharmacological Research
2CNR, Institute of Neuroscience, Cellular and Molecular
Pharmacology Section, Milan, Italy
3Dompé Research Center, L’Aquila, Italy
4Amgen Inc., Thousand Oaks, California, USA
Introduction
There have been reports that granulocyte-colony stimulating factor
(G-CSF) has anti-inflammatory properties in various animal models,
including those for experimental colitis [1] and
superantigen-induced shock [2]. More importantly, it has been
reported that G-CSF-mobilized peripheral stem cell transplantation
reduces the severity of acute graft-versus-host disease (GvHD)
[3-5]. However, treatment of recipients with G-CSF after bone
marrow transplantation has been associated with an increased
incidence of GvDH [6, 7].
Administration of G-CSF to humans decreases inflammatory
cytokine production, particularly tumor necrosis factor (TNF), by
whole blood or dendritic cells ex vivo [5, 8-11], while it
increases the production of cytokine antagonists such as
interleukin-1 receptor antagonist (IL-1ra) and soluble TNF
receptors [8, 9].
The aim of this work was to investigate the mechanism for the
G-CSF-induced decrease of TNF production, ex vivo, using mice. In
particular, we focused our attention on the role of endogenous
mediators known to inhibit TNF production, interleukin-10 (IL-10)
and prostaglandins. Our results indicate that G-CSF increases the
production of IL-10 and prostaglandin E2
(PGE2), which in turn mediate the inhibition of
lipopolysaccharide (LPS)-induced TNF production.
Methods
Animals and treatments
Male CD-1 mice (20-25 g body weight) were purchased from Charles
River (Calco, Italy). Procedures involving animals and their care
were conducted in conformity with the institutional guidelines that
are in compliance with national (D.L. n° 116, G.U., suppl. 40, 18
Febbraio 1992, Circolare n° 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, U.S.
National Research Council, 1996) laws and policies. Mice were given
100 μg/kg s.c. of recombinant murine G-CSF (Amgen, Thousand Oaks,
CA, USA) dissolved in saline, either once, one hour before
bleeding, or daily for three consecutive days, and blood was
withdrawn 24 hours after the last treatment. Control mice were
given saline alone. Mice were bled by puncture of the retro-orbital
plexus under ether anesthesia, and blood was collected over heparin
(14 U/mL; Liquemin, Roche, Milan, Italy).
LPS-induced cytokine production in murine whole blood
Blood was then plated onto 96-well tissue culture plates (100
μL/well) and incubated for four hours at 37 °C in a 5%
CO2, humidified atmosphere, with or without 1 μg/mL of
LPS (Escherichia coli 055:B5, Sigma, St. Louis, MO, USA). In some
experiments, blood was stimulated in the presence of 1 μM of
ketoprofen (lysine salt, Dompé, L’Aquila, Italy) and/or 200 μg/mL
of JES5-2A5 monoclonal antibody, a rat IgG1 neutralizing mouse
IL-10 (a kind gift from T. Mosmann, University of Alberta,
Edmonton, Canada), which were added to culture plates 15 min before
LPS. At the end of the incubation, blood was diluted 1:1 (v/v) with
ice-cold saline, centrifuged, and the supernatants were collected
for TNF, IL-10 and PGE2 determination.
TNF activity was measured by the degree of cytotoxicity on L929
cells, in the presence of 1 μg/mL actinomycin D, as previously
described [12], using recombinant TNF as standard. The sensitivity
of this assay is approximately 0.33 U/mL. PGE2 and IL-10
were measured by EIA using commercially available kits from
Amersham (Little Chalfont, UK) and BioSource International
(Camarillo, CA, USA), respectively.
Results
As reported in table 1( Table 1 ), a
three-day G-CSF pretreatment led to a marked increase in neutrophil
(27-fold), monocyte (five-fold) and lymphocyte (two-fold) counts in
peripheral blood 24 hours after the last treatment. In contrast, a
single G-CSF pretreatment, one hour before bleeding, brought about
only a small increase in the neutrophil count (G-CSF, 2.0±1.3 x
106/mL versus saline, 1.0±0.5 x 106/mL), with
no difference in monocyte and lymphocyte counts.
We measured the production of TNF in whole blood obtained from
G-CSF-treated mice after ex vivo stimulation with LPS. As shown in
( Fig. 1 )
(left), TNF production was markedly reduced (by about 95%) in blood
from mice pretreated with G-CSF daily for three days in comparison
with saline-treated control mice. However, when mice were given a
single dose of G-CSF, one hour before blood sampling, there was no
significant change in TNF production (( Fig. 1 ), right). No TNF
was detectable in unstimulated blood (data not shown).
We then evaluated the production of two well-known inhibitors of
TNF synthesis, IL-10 and PGE2, in the blood of mice
pretreated with G-CSF for three days. As shown in ( Fig. 2 ), the levels of
both PGE2 and IL-10 were similar in blood from both
G-CSF and saline-treated mice in the absence of stimulation.
However, LPS stimulation induced a two-fold increase in
PGE2 concentration and a 10-fold increase in IL-10
concentration in blood from G-CSF-treated mice in comparison with
unstimulated blood. Nevertheless, we did not find any significant
increase in either PGE2 or in IL-10 levels in blood from
saline-treated mice after LPS stimulation (( Fig. 2 )).
We also investigated whether G-CSF, added in vitro to whole
blood from untreated mice at times up to one hour before addition
of LPS and at concentrations ranging from 10 ng/mL to 10 μg/mL,
could modulate LPS-induced TNF or IL-10 production, however we did
not observe any significant effect (data not shown).
In order to investigate the role of prostaglandins and/or IL-10
in the decrease in TNF production induced by G-CSF pretreatment, we
performed the blood stimulation in the presence of the
cyclooxygenase (COX) inhibitor, ketoprofen, and a neutralizing
anti-IL-10 antibody. We found that the IL-10 produced by whole
blood under our experimental conditions was completely neutralized
by the addition of the monoclonal antibody (data not shown);
likewise, ketoprofen completely inhibited LPS-induced
PGE2 production in blood from G-CSF-treated mice (data
not shown). As shown in ( Fig. 3 ), ketoprofen alone
did not reverse the inhibition of TNF production by G-CSF, whereas
the anti-IL-10 antibody significantly reversed this inhibition
(from 94 % to 47 %). Notably, combined addition of ketoprofen and
anti-IL-10 antibody completely restored TNF production. Similar
results were obtained when indomethacin was used as the COX
inhibitor instead of ketoprofen (data not shown).
Table 1 Changes in peripheral blood leukocyte counts
after a three-day G-CSF pretreatment
|
Saline
|
G-CSF
|
|
Neutrophils (X 106/mL)
|
1.1 ± 0.4
|
29.8 ± 6.3 **
|
|
Monocytes (X 106/mL)
|
0.2 ± 0.05
|
0.8 ± 0.4 **
|
|
Lymphocytes (X 106/mL)
|
5.7 ± 1.3
|
14.0 ± 2.1 **
|
Discussion
The results reported here demonstrate that G-CSF inhibits the
ability of blood cells to produce TNF by increasing the production
of IL-10 and PGE2. It is important to note that these
are not direct effects of G-CSF. In fact, neither short-term
pretreatment with G-CSF for one hour nor addition of G-CSF in vitro
to whole blood in culture, had any significant effect on TNF
production. Since one hour pretreatment impacted leukocyte counts
only minimally, while three days pretreatment had a marked effect,
it is possible that the increased production of IL-10 and
PGE2 induced by G-CSF is a consequence of the changes in
cell populations brought about the three-day G-CSF pretreatment.
Similar studies performed in humans reported that
G-CSF-mobilized peripheral blood mononuclear cells (PBMC) display a
decreased cell proliferation in response to alloantigen, in
comparison with control PBMC [13]. This hyporesponsiveness has been
ascribed to an increased production of IL-10 by CD14+
monocytes in G-CSF-treated patients [14].
The importance of IL-10 and PGE2 as negative feedback
regulators of TNF production has been widely documented in reports
showing that TNF production, both in vivo and in vitro, is
increased by anti-inflammatory drugs acting as COX inhibitors [15,
16] and by inhibition of IL-10 using antibodies or knock-out mice
[17-19].
At first it may seem surprising to consider PGE2 as
an anti-TNF mediator, since prostaglandins are well known
inflammatory molecules. On the other hand, the paradoxical increase
of TNF production by non-steroidal anti-inflammatory drugs has been
extensively documented in animals [20, 21] and in human volunteers
[22]. Furthermore, while evaluating the inflammatory versus
anti-inflammatory (anti-TNF) activities of PGE2, it is
important to consider the relative contribution of PGE2
and IL-10 to the TNF inhibitory effect of G-CSF. In fact, in our
experimental model, IL-10 seems to be far more important than
PGE2 as an inhibitor of TNF production, on two counts:
1) G-CSF pretreatment increased IL-10 production by a factor
of 10, while the production of PGE2 was increased only
by a factor of two; and 2) the anti-IL-10 antibody alone
markedly reversed inhibition of TNF production by G-CSF, while
ketoprofen had no effect alone and only showed effects in
combination with the anti-IL-10 antibody.
In conclusion, our data provide additional information for the
interactions between IL-10 and PGE2, which should not be
overlooked when considering the role of these mediators as feedback
inhibitors. In particular, it is known that while IL-10 inhibits
prostaglandin production, which contributes to the antiinflammatory
action of IL-10, [23, 24], PGE2 induces IL-10 production
[24, 25], an observation that fits well with the negative feedback
described in the present paper.
If one considers that increased IL-10 production was observed in
G-CSF-mobilized mononuclear cells [14], it is possible that the
mechanism elucidated in our animal model might explain the
inhibitory action of G-CSF on TNF production in humans.
References
1 Hommes DW, Meenan J, Dijkhuizen S, Ten
Kate FJ, Tytgat GN, Van Deventer SJ. Efficacy of
recombinant granulocyte colony-stimulating factor (rhG-CSF) in
experimental colitis. Clin Exp Immunol 1996; 106: 529.
2 Aoki Y, Hiromatsu K, Kobayashi N, et al.
Protective effect of granulocyte colony-stimulating factor against
T-cell-meditated lethal shock triggered by superantigens. Blood
1995; 86: 1420.
3 Zeng D, Dejbakhsh-Jones S, Strober S.
Granulocyte colony-stimulating factor reduces the capacity of blood
mononuclear cells to induce graft-versus-host disease: impact on
blood progenitor cell transplantation. Blood 1997; 90: 453.
4 Pan L, Teshima T, Hill GR, et al.
Granulocyte colony-stimulating factor-mobilized allogeneic stem
cell transplantation maintains graft-versus-leukemia effects
through a perforin-dependent pathway while preventing
graft-versus-host disease. Blood 1999; 93: 4071.
5 Reddy V, Hill GR, Pan L, et al. G-CSF
modulates cytokine profile of dendritic cells and decreases acute
graft-versus-host disease through effects on the donor rather than
the recipient. Transplantation 2000; 69: 691.
6 Remberger M, Naseh N, Aschan J, et al.
G-CSF given after haematopoietic stem cell transplantation using
HLA-identical sibling donors is associated to a higher incidence of
acute GVHD II-IV. Bone Marrow Transplant 2003; 32: 217.
7 Ringden O, Labopin M, Gorin NC, et al.
Treatment with granulocyte colony-stimulating factor after
allogeneic bone marrow transplantation for acute leukemia increases
the risk of graft-versus-host disease and death: a study from the
Acute Leukemia Working Party of the European Group for Blood and
Marrow Transplantation. J Clin Oncol 2004; 22: 416.
8 Hartung T, Docke WD, Gantner F, et al.
Effect of granulocyte colony-stimulating factor treatment on ex
vivo blood cytokine response in human volunteers. Blood 1995; 85:
2482.
9 Dalhoff K, Hansen F, Dromann D, Schaaf B,
Aries SP, Braun J. Inhibition of neutrophil apoptosis and
modulation of the inflammatory response by granulocyte
colony-stimulating factor in healthy and ethanol-treated human
volunteers. J Infect Dis 1998; 178: 891.
10 Boneberg EM, Hareng L, Gantner F,
Wendel A, Hartung T. Human monocytes express functional
receptors for granulocyte colony-stimulating factor that mediate
suppression of monokines and interferon-gamma. Blood 2000; 95:
270.
11 Hartung T, Doecke WD, Bundschuh D, et al.
Effect of filgrastim treatment on inflammatory cytokines and
lymphocyte functions. Clin Pharmacol Ther 1999; 66: 415.
12 Aggarwal BB, Kohr WJ, Hass PE, et al.
Human tumor necrosis factor. Production, purification, and
characterization. J Biol Chem 1985; 260: 2345.
13 Mielcarek M, Martin PJ, Torok-Storb B.
Suppression of alloantigen-induced T-cell proliferation by CD14+
cells derived from granulocyte colony-stimulating factor-mobilized
peripheral blood mononuclear cells. Blood 1997; 89: 1629.
14 Mielcarek M, Graf L, Johnson G,
Torok-Storb B. Production of interleukin-10 by granulocyte
colony-stimulating factor-mobilized blood products: a mechanism for
monocyte-mediated suppression of T-cell proliferation. Blood 1998;
92: 215.
15 Kunkel SL, Spengler M, May MA,
Spengler R, Larrick J, Remick D. Prostaglandin E2
regulates macrophage-derived tumor necrosis factor gene expression.
J Biol Chem 1988; 263: 5380.
16 Renz H, Gong JH, Schmidt A, Nain M,
Gemsa D. Release of tumor necrosis factor-alpha from
macrophages. Enhancement and suppression are dose-dependently
regulated by prostaglandin E2 and cyclic nucleotides. J Immunol
1988; 141: 2388.
17 de Waal Malefyt R, Abrams J, Bennett B,
Figdor CG, de Vries JE. Interleukin 10(IL-10) inhibits
cytokine synthesis by human monocytes: an autoregulatory role of
IL-10 produced by monocytes. J Exp Med 1991; 174: 1209.
18 Berg DJ, Kuhn R, Rajewsky K, et al.
Interleukin-10 is a central regulator of the response to LPS in
murine models of endotoxic shock and the Shwartzman reaction but
not endotoxin tolerance. J Clin Invest 1995; 96: 2339.
19 Agnello D, Villa P, Ghezzi P. Increased tumor
necrosis factor and interleukin-6 production in the central nervous
system of interleukin-10-deficient mice. Brain Res 2000; 869:
241.
20 Pettipher ER, Wimberly DJ. Cyclooxygenase
inhibitors enhance tumour necrosis factor production and mortality
in murine endotoxic shock. Cytokine 1994; 6: 500.
21 Sironi M, Gadina M, Kankova M, et al.
Differential sensitivity of in vivo TNF and IL-6 production to
modulation by anti-inflammatory drugs in mice. Int J
Immunopharmacol 1992; 14: 1045.
22 Martich GD, Danner RL, Ceska M,
Suffredini AF. Detection of interleukin 8 and tumor necrosis
factor in normal humans after intravenous endotoxin: the effect of
antiinflammatory agents. J Exp Med 1991; 173: 1021.
23 Berg DJ, Zhang J, Lauricella DM,
Moore SA. Il-10 is a central regulator of cyclooxygenase-2
expression and prostaglandin production. J Immunol 2001; 166:
2674.
24 Niiro H, Otsuka T, Kuga S, et al. IL-10
inhibits prostaglandin E2 production by
lipopolysaccharide-stimulated monocytes. Int Immunol 1994; 6:
661.
25 Kambayashi T, Alexander HR, Fong M,
Strassmann G. Potential involvement of IL-10 in suppressing
tumor-associated macrophages. Colon-26-derived prostaglandin E2
inhibits TNF-alpha release via a mechanism involving IL-10. J
Immunol 1995; 154: 3383.
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