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Departments of
*
Internal Medicine and
Pathology, University of Iowa College of Medicine, Iowa City, IA 52242
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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, TNF-
, or IL-12 markedly decreased the
induction of COX-2 in IL-10-/- spleen cells, suggesting
that increased inflammatory cytokine production mediates much of the
COX-2 induction in IL-10-/- mice. Treatment of
IL-10-/- mice with low doses of LPS resulted in a marked
induction of COX-2 mRNA in the spleen, whereas wt mice had minimal
expression of COX-2 mRNA. These findings indicate that, in addition to
IL-10’s central role in the regulation of inflammatory cytokines,
endogenous IL-10 is an important regulator of PG production in the
response to LPS. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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PG biosynthesis is tightly controlled. The key regulatory enzyme of the
PG biosynthesis pathway, PG synthase (EC 1.14.99.1), also known as
cyclooxygenase (COX),3
is the first enzyme in the biosynthetic pathway leading to PG,
thromboxanes, and prostacyclins. The COX enzyme exists in two isoforms:
COX-1, a constitutive form that is expressed in multiple cell types and
is thought to produce PGs central to physiologic homeostasis
(7); and COX-2, an inducible form that is rapidly
up-regulated in response to LPS, cytokines, and mitogens
(7, 8, 9). LPS is a potent inducer of PG synthesis. It has
been demonstrated that LPS-stimulated monocytes rapidly induce COX-2 at
both the mRNA and protein level (10, 11, 12). Multiple
cytokines have been demonstrated to alter COX-2 expression in vitro.
Proinflammatory cytokines including TNF- (13, 14),
IL-1 (14, 15), and IFN- (16, 17) have
been demonstrated to induce COX-2 expression, whereas
anti-inflammatory cytokines such as IL-4 (18), IL-13
(19, 20), and IL-10 (21) can inhibit COX-2
induction. The pleiotropic cytokine TGF-
can enhance
(22) or inhibit (23) COX-2 expression,
depending upon the cell type tested. The functional significance of
these in vitro observations has not been established in vivo.
IL-10 is a cytokine with potent anti-inflammatory activity. IL-10
has been shown to be a potent macrophage deactivator, blocking the
induced synthesis of TNF-, IL-1, IL-6, IL-8, and GM-CSF by human
monocytes (24) and mouse peritoneal macrophages
(25). IL-10 also indirectly suppresses the synthesis of
IFN- by Th cells (26) and NK cells (27).
Interestingly, IL-10 was found to be produced by monocytes after
exposure to LPS (24), which suggested that IL-10 may
regulate the inflammatory response to LPS. Indeed, we found that
LPS-induced cytokine production was markedly increased in
IL-10-/- mice and that the lethal dose of LPS
for IL-10-/- mice was 20-fold lower than that
for wild-type (wt) mice. These data demonstrate that endogenous
production of IL-10 is a central regulator of cytokine production in
response to LPS (28). It has recently been reported that
IL-10 in vitro can inhibit COX-2 induction in human monocytes and
neutrophils (18, 21). These studies suggested that IL-10
may regulate the production of lipid mediators.
The generation of mice with a targeted disruption of the IL-10 gene (IL-10-/-) (29) has allowed further definition of the actions of IL-10 in complex physiologic systems. We have compared LPS-induced COX expression and PG production in IL-10-/- and wt mice to determine the importance of endogenous IL-10 in the regulation of PG production.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Healthy 5- to 6-wk-old IL-10-/- mice on a 129/SvEv background were used for this study (30). Wild-type 129/SvEv mice were obtained from Taconic Farms (Germantown, NY). Mice were maintained in microisolator cages under specific pathogen-free conditions at the animal care facility at the University of Iowa.
Reagents
LPS from Escherichia coli (serotype 0111:B4) was
obtained from Difco (Detroit, MI) and resuspended in pyrogen-free
saline. Aminoguanidine (hemisulfate salt) and A23187 were obtained from
Sigma (St. Louis, MO). NS-398 was obtained from Cayman Chemicals (Ann
Arbor, MI). Neutralizing Ab to IL-10 (JESS-2A5) and recombinant IL-10
(expressed in E. coli) were kind gifts of D. Rennick (DNAX,
Palo Alto, CA). Rabbit polyclonal anti-murine COX-2 was obtained
from Cayman Chemicals; and rabbit polyclonal anti-COX-1 was
obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-cytokine
Abs (anti-TNF-, XT22; anti-IFN-, XMG-1.2; anti-IL-12,
C17-8; and isotype control Ab, MOPC-21) were kind gifts of J. Weinstock
(University of Iowa).
HPLC
For the HPLC assay of eicosanoids, spleen cell cultures were
incubated for 30 min at 37°C with up to 7.5 µM
[5,6,8,9,11,12,14,15-3H]arachidonic acid (AA)
(155 Ci/mmol; New England Nuclear, Boston, MA) in serum-free medium.
After incubation, the medium was collected and centrifuged to remove
cellular debris. In some experiments, after incubation with
[3H]AA, the medium was removed and replaced
with serum-free medium containing the calcium ionophore A23187 (2
µM). Lipids were then extracted with a 2:1 (v/v) mixture of
chloroform/methanol containing 1% acetic acid, dried under
N2 and resuspended in acetonitrile. Lipids were
separated on a 332 HPLC system (Beckman Coulter, Fullerton, CA)
equipped with a C18 reverse-phase column containing 5-µm spherical
packing with the use of a gradient of water (adjusted to pH 3.5 with
phosphoric acid) and acetonitrile. The column effluent was mixed with
scintillation solution at a 1:3 ratio before passing through an on-line
radioactivity detector (Flo-1; Radiomatic Instruments and Chemicals,
Tampa, FL). The system was standardized with the following eicosanoids:
6-keto-PGF1, PGF2,
PGE2, leukotriene B4
(LTB4), and AA (Amersham Pharmacia Biotech,
Piscataway, NJ). Control studies performed with empty culture dishes
demonstrated that >95% of the added radioactivity remained unchanged
and that no appreciable nonenzymatic modifications occurred during
these incubations or extractions.
Cell culture protocols
Spleen cells from wt or IL-10-/- mice were cultured at 5 x 106 cells/ml in RPMI 1640 supplemented with 10% FCS, 2 mM L-glutamine, 0.05 mM 2-ME, penicillin (100 U/ml), and streptomycin (100 U/ml) in 12-well tissue culture plates (Costar, Corning, NY). Cells were incubated in medium alone or medium supplemented with LPS at 10 µg/ml; this concentration of LPS gave maximal stimulation of PG production from both wt and IL-10-/- spleen cell cultures. Supernatants from triplicate cultures were harvested after 24 h and stored at -70°C before analysis for PG concentration. Spleen cells were subsequently harvested for either RNA or protein isolation. In some cultures, after 24 h of LPS stimulation the supernatant was removed, cells were incubated in PBS for 30 min in the presence or absence of NS-398 (10 µM), and the supernatant was subsequently assayed for PGE2 production.
To assess the role of inflammatory cytokines in the induction of COX-2,
LPS-stimulated spleen cells from IL-10-/- mice
were incubated in the presence of anti-cytokine Abs
(anti-TNF-, XT22, 2.5 µg/ml; anti-IFN-, XMG-1.2, 5.0
µg/ml; or anti-IL-12, C17-8, 3.0 µg/ml) or isotype control Ab
(MOPC-21, 5.0 µg/ml).
Quantification of PGE2
PGE2 levels in tissue culture supernatants were determined using the PGE2 enzyme immunoassay kit (Cayman Chemicals) according to the manufacturer’s instructions.
Northern blotting
RNA from spleens or spleen cell cultures was prepared using RNA-STAT-60 (Tel-Test, Friendswood, TX) exactly as per the manufacturer’s instructions. Total RNA (15 µg/lane) was electrophoresed through a 1.0% agarose-4% formaldehyde gel and transferred onto a nylon membrane. cDNA fragments specific for murine COX-1 and COX-2 (Oxford Biomedical, Oxford, MI) were 32P-labeled by the random prime method and used for sequential hybridizations. Membrane hybridization was conducted in 50% formamide at 42°C overnight with 2 x 106 cpm/ml cDNA probe. After a series of washings in 2x SSC at room temperature, in 0.2x SSC, 0.1% SDS at 60°C, and in 0.1x SSC at room temperature, the membranes were exposed to Fuji RX film (Tokyo, Japan) at -70°C. Ethidium bromide staining of 18S and 28S ribosomal RNA served as an internal loading control.
Western blotting
Protein was isolated from cultured spleen cells by resuspending in lysis buffer (50 mM Tris (pH 7.5), 150 mM NaCl, 100 µg/ml PMSF, 1 µg/ml aprotinin, 1 µg/ml leupeptin, 1 mM diethyldithiocarbamic acid, 1% Nonidet P-40, and 1% sodium deoxycholate). Cells were lysed by sonication (20 s, 4°C). Debris was eliminated by centrifugation (15 min, 1000 x g). Protein concentration was measured using a commercial reagent based on bicinchoninic acid staining (Pierce, Rockford, IL) using BSA as an internal standard. Equal amounts of cellular protein were loaded onto a 10% polyacrylamide gel and separated by electrophoresis (200 V for 45 min). Proteins were then transferred to nitrocellulose (100 V for 1 h), and the membrane was blocked with 5% nonfat dry milk. The nitrocellulose was then incubated with a rabbit polyclonal primary Ab (anti-COX-2, 1:1000; Cayman Chemicals; anti-COX-1, 1:1000; Santa Cruz Biotechnology) overnight at 25°C. Ab labeling was detected using ECL (Amersham) as per the manufacturer’s instructions. Specificity of the anti-COX Ab was confirmed with the use of ram seminal vesicle COX-1 (Oxford Biomedical) and sheep placenta COX-2 (Cayman Chemicals).
Films and photographs of Northern or Western blots were scanned in at 600 dpi using an Epson Expression 1600 scanner. Densitometric analysis was performed using Vtrace (developed at the University of Iowa Image Analysis Facility) operating on a Silicon Graphics O2 workstation (Mountain View, CA). Average and integrated OD measurements were made on user-selected regions. A Kodak photographic step tablet (Rochester, NY) was used to calibrate OD.
RNase protection assay
A murine COX-2 cDNA fragment (nt-205–505; GenBank accession no. M88242) was synthesized by RT-PCR using mouse brain RNA as a template and cloned into pGEM-4. Fragments of the RPL32-4A gene (31) and the inducible NO synthase (iNOS) gene (32) were also cloned into pGEM-4. L32 served as an internal loading control. RNase protection assay for the detection of COX-2 and iNOS was performed as previously described (33). Briefly, for the synthesis of a 32P-radiolabeled antisense RNA probe, equimolar mixtures of the linearized COX-2, iNOS, and L32 templates were used. Hybridization reactions were performed overnight at 56°C. Following RNase digestion, the RNA duplexes were isolated by electrophoresis in a standard 7.5% acrylamide/12 M urea/0.5% Tris/borate/EDTA sequencing gel. Dried gels were placed on BMR film and were exposed at -70°C.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The profile of eicosanoids produced by spleen cells from labeled
AA was characterized to assess whether endogenous IL-10 regulates the
types of eicosanoids produced in response to LPS. Spleen cells from wt
or IL-10-/- mice were incubated with LPS (10
µg/ml) or medium alone for 24 or 48 h and subsequently exposed
to 7.5 µM [3H]AA. The released
[3H]AA metabolites were analyzed using
reverse-phase HPLC (Fig. 1
). Based on the
retention time, the most prominent eicosanoid produced by the spleen
cell cultures was PGE2. Under these culture
conditions, spleen cells from wt mice did not produce significant
amounts of PGs or leukotrienes even when stimulated with LPS (Fig. 1, A and B). In contrast, incubation of
IL-10-/- spleen cells for 48 h in medium
alone resulted in the production of small amounts of
PGE2 as well as LTB4 (Fig. 1C). When stimulated with LPS,
IL-10-/- spleen cells produced more
PGE2, while not appreciably changing their
release of LTB4 (Fig. 1D).
Qualitatively similar results were obtained when spleen cells were
incubated with LPS for 24 h. Stimulation of the
3H-labeled cells with the calcium ionophore
A23187 (2 µM) produced a qualitatively similar eicosanoid profile
(data not shown).
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). Spleen cells from LPS-stimulated wt
mice also produced PGE2, albeit in smaller
amounts than the IL-10-/- spleen cells. To
assess PGE2 production, wt and
IL-10-/- spleen cell cultures were incubated
with LPS for 24 h. Subsequently, the medium was replaced with PBS
for 30 min, and the supernatant was assayed for
PGE2. PGE2 production was
markedly up-regulated in the IL-10-/- spleen
cells as compared with wt (Fig. 3).
Incubation of the cells with NS-398, a COX-2 selective inhibitor,
blocked PGE2 production, demonstrating that the
PGE2 production was derived from the COX-2
enzyme.
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Because we had found that LPS stimulation markedly increased PG
production from IL-10-/- spleen cells, we next
determined whether endogenous IL-10 would regulate the expression of
COX-2 mRNA. Spleen cells from wt or IL-10-/-
mice were incubated in medium or LPS (10 µg/ml) for varying time
points, and COX-2 mRNA levels were subsequently determined. As
demonstrated in Fig. 4, no COX-2 mRNA was
seen in unstimulated wt and IL-10-/- spleen
cells. Within 2 h of LPS stimulation, COX-2 mRNA levels increased
in both wt and IL-10-/- spleen cells; however,
at 24 h the magnitude of increase was 6.3-fold greater in the
IL-10-/- cells than in the wt cells (Fig. 4).
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We next assessed whether endogenous IL-10 would alter the level of
protein expression of COX-1 and COX-2. Stimulation of
IL-10-/- spleen cells with LPS resulted in a
marked induction of COX-2 protein as compared with wt spleen cells
(Fig. 5A). In contrast, the
absence of IL-10 had no reproducible effect on the level of protein
expression of COX-1 (Fig. 5B). Wild-type spleen cells were
stimulated with LPS in the presence of neutralizing Abs to IL-10 to
further assess the role of endogenous IL-10 in COX-2 expression.
Neutralization of IL-10 resulted in a 1.8-fold increase in COX-2
protein expression (Fig. 5C).
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Inflammatory cytokines such as TNF- are known to be potent
inducers of COX-2 expression (34, 35). Because
IL-10-/- mice markedly overproduce inflammatory
cytokines in response to LPS (29), we tested whether the
marked induction of COX-2 mRNA and protein in
IL-10-/- spleen cells was secondary to
increased production of inflammatory cytokines. Spleen cells from
IL-10-/- mice were incubated with LPS in the
presence or absence of neutralizing Ab to TNF-, IFN-, or IL-12,
and the induction of COX-2 protein was assessed by Western blotting
(Fig. 6). Neutralization of IFN- and
TNF- resulted in significant decreases in LPS-induced COX-2
expression (IFN-, 64.0 ± 16.5%; TNF-, 75 ± 5.57%;
mean percent decrease ± SD). Neutralization of IL-12 also
resulted in a significant decrease in protein expression of COX-2
(49 ± 15.8%). As reported previously (21),
incubation of spleen cells with IL-10 (10 U/ml) inhibited LPS induction
of COX-2 protein (data not shown).
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To ascertain whether endogenous IL-10 would regulate the
expression of COX-2 in vivo, wt and IL-10-/-
mice were treated with a low dose of LPS (25 µg, i.p.), and splenic
mRNA expression of COX-2 was assessed by RNase protection (Fig. 7). The induction of COX-2 mRNA in
IL-10-/- mice was markedly greater than that of
wt mice. COX-2 mRNA was not detected in control wt or
IL-10-/- mice. Interestingly, iNOS mRNA was
also markedly up-regulated in the IL-10-/-
mice, potentially increasing the production of NO, which may
up-regulate COX-2 enzyme activity (36).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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PGs are potent bioactive lipid mediators, and thus PG production is tightly controlled. The increased PG production observed in IL-10-/- spleen cells in response to LPS occurs together with markedly increased levels of mRNA and protein for the inducible form of COX, COX-2. This finding is consistent with previous studies that demonstrated that PG production in response to LPS, inflammatory cytokines, and/or mitogens is due almost entirely to the rapid induction of the COX-2 isoform (10, 11). Our finding that NS-398, a selective COX-2 inhibitor, blocks the increased PG production from IL-10-/- spleen cells confirms that the increased PG production in our system was secondary to increased COX-2 expression and/or activity. In contrast, IL-10 appeared to exert no regulatory effect on COX-1 protein expression, similar to previous reports indicating that COX-1 expression is generally constitutive rather than inducible (37).
IL-10 may regulate COX-2 mRNA expression through either indirect or
direct means. Direct regulation of COX-2 by IL-10 may occur at the
transcriptional level via regulation of NF-
B. The COX-2 promoter
contains two NF-B motifs (38) that are clearly involved
in the regulation of COX-2 expression in both mouse (39)
and human (40) macrophage cell lines. IL-10 has been shown
to inhibit inflammatory cytokine production via inhibition of NF-B
function (41). Therefore, the absence of IL-10 in the
IL-10-/- mouse may have led to enhanced NF-B
activity, resulting in increased levels of COX-2 mRNA.
IL-10 may regulate COX-2 gene expression at the posttranscriptional
level as well. One of the mechanisms by which IL-10 suppresses
inflammation is via induction of instability in the mRNAs of
inflammatory cytokines, resulting in their degradation. The
mRNAs for multiple inflammatory cytokines (TNF-, IL-1,
IL-1, GM-CSF) and chemokines (KC, macrophage inflammatory protein
1, macrophage inflammatory protein 1) all contain AU-rich
elements in their 3' untranslated regions, a sequence that has been
found to be important for mRNA stability and translation (42, 43). IL-10 has been reported to destabilize the mRNA for all of
these cytokines (44). The 3' untranslated region of COX-2
mRNA has 20 copies of the pentamer AUUUA (38), suggesting
that COX-2 mRNA expression may be regulated posttranscriptionally.
Indeed, LPS and IL-1 enhance COX-2 mRNA stability (45),
whereas exogenous IL-10 in vitro accelerates the degradation of COX-2
mRNA in human monocytes (21). Therefore, the absence of
IL-10 may have resulted in increased COX-2 mRNA through an increase in
the mRNA stability.
Our study indicates that IL-10 may regulate COX-2 expression and PG
production indirectly via regulation of proinflammatory cytokines.
COX-2 mRNA is known to be induced by multiple cytokines including
TNF- and IL-1 (46, 47). In constrast, IL-10 is an
important down-regulator of inflammatory cytokines (for example,
TNF- and IL-1) from LPS-stimulated macrophages (25).
We have previously demonstrated that LPS stimulation of
IL-10-/- mice in vivo results in markedly
increased and sustained production of TNF-, IFN-, and IL-12
(28). Our findings clearly demonstrate that neutralization
of TNF- and IFN- resulted in a marked decrease in COX-2 induction
in LPS-stimulated IL-10-/- cells.
Interestingly, this study demonstrated that neutralization of IL-12
resulted in a significant decrease in COX-2 expression as well. IL-12
is produced by macrophages, neutrophils, and dendritic cells in
response to infection or bacterial products such as LPS (48, 49). Ligation of the IL-12 receptor that is expressed on T cells
and NK cells promotes the development of IFN--producing Th1
CD4+ T cells and induces high-level production of
IFN- (49). In mouse macrophages, IFN- enhances the
effect of LPS on COX-2 expression (50). In contrast, IL-10
is a potent inhibitor of IL-12 production (51), and
LPS-treated IL-10-/- mice markedly over-produce
IL-12 (28). Therefore, neutralization of IL-12 may have
indirectly decreased COX-2 production due to decreased IFN-
production. Taken together, these results indicate that much of the
LPS-induced increase in COX-2 expression in
IL-10-/- mice is secondary to dysregulated
inflammatory cytokine production in these mice.
The relationship between NO production and PG biosynthesis is quite
complex. NO donors have been reported to stimulate or inhibit PG
production in a variety of systems (36, 52, 53, 54, 55). It has
recently been reported that iNOS-deficient mice have markedly decreased
PGE2 production in response to LPS and IFN-,
clearly supporting the view that NO and/or NO-derived species modulate
COX activity and PG production (56). In our in vitro
studies, we did not observe increased NO production from LPS-stimulated
IL-10-/- spleen cell cultures at 24 h
(data not shown), whereas PGE2 production at this
time point was clearly elevated, suggesting that NO production was not
central to the increased PGE2 production from
IL-10-/- spleen cells. Interestingly, in vivo
we found marked induction of iNOS mRNA in response to LPS in the
IL-10-/- mice, consistent with our previous
study in which we demonstrated that IL-10-/-
mice produce excessive amounts of NO in vivo in response to LPS
(28). The kinetics of iNOS induction and NO production in
vivo may differ significantly from that in vitro and it is quite
possible that NO or NO-derived species such as peroxynitrite have
important effects on in vivo production of PGs in
IL-10-/- mice. Further studies will be required
to resolve this issue.
This study clearly demonstrates that endogenously produced IL-10 is a
key regulator of COX-2 expression and subsequent PG production. The
regulatory effect of IL-10 on PG production may be both direct (via
inhibition of mRNA expression and decreasing the half-life of the COX-2
mRNA) and indirect (via regulation of inflammatory cytokines that can
induce COX-2 expression). PGs are potent lipid mediators of the immune
and inflammatory response; however, their role is quite complex. For
example, PGE2 appears to be a key mediator of the
inflammatory response: in an animal model of arthritis, neutralization
of PGE2 resulted in decreased edema,
hyperalgesia, and IL-6 production (3). However,
PGE2 may have anti-inflammatory effects:
PGE2 in vitro can inhibit cytokine production,
including IL-12 production and function (57, 58, 59) and
TNF- production (60, 61). PGs may also regulate T cell
differentiation and function (59, 62), favoring the
production of Th2 T cells over proinflammatory Th1 T cells. We
previously reported that IL-10-/- mice have
exaggerated inflammatory responses (28, 63) as well as
dysregulated mucosal immune responses (30, 64)
characterized by increased expression of peptide mediators. The results
of the studies described herein suggest that increased PG production is
also a major component of the altered immune and inflammatory responses
seen in IL-10-/- mice. Further studies will be
required to determine the precise role played by PGs in the altered
immune/inflammatory response in IL-10-/-
mice.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Daniel J. Berg, University of Iowa Hospitals, Department of Internal Medicine, C32-GH, 200 Hawkins Drive, Iowa City, IA 52242.
3 Abbreviations used in this paper: COX, cyclooxygenase; wt, wild type; LTB4 leukotriene B4; AA, arachidonic acid; iNOS, inducible NO synthase.
Received for publication July 21, 2000. Accepted for publication November 22, 2000.
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M. M. Monick, P. K. Robeff, N. S. Butler, D. M. Flaherty, A. B. Carter, M. W. Peterson, and G. W. Hunninghake Phosphatidylinositol 3-Kinase Activity Negatively Regulates Stability of Cyclooxygenase 2 mRNA J. Biol. Chem., August 30, 2002; 277(36): 32992 - 33000. [Abstract] [Full Text] [PDF]
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