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Bristol-Myers Squibb Pharmaceutical Research Institute, Seattle, WA 98121; and
Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, NJ 08543
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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and lethality in a dose-dependent
manner. Consistent with these findings, OM potently suppressed
inflammation and tissue destruction in murine models of rheumatoid
arthritis and multiple sclerosis. T cell function and Ab production
were not impaired by OM treatment. Taken together these data indicate
the activities of this cytokine in vivo are antiinflammatory without
concordant immunosuppression. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The exact participation of each cytokine in the inflammatory disease
process is poorly understood in part due to their complex interplay.
However, the ability of a variety of cytokine and cytokine agonists to
alter the severity or course of various inflammatory diseases is an
impressive testament to the clinical value of cytokines as a target for
therapeutic intervention (2, 3). Such data has been accrued using
animal models of disease, transgenic animals and, more recently,
clinical trials of cytokine inhibitors (4, 5). A variety of approaches
are currently being studied to alter cytokine function to bring about
the regulation of aberrant inflammatory responses (6). Inhibitors of
proinflammatory cytokines, most notably TNF- inhibitors, have been
successful in moderating untoward inflammatory responses (2). Abs to
TNF- and soluble receptors are currently in clinical trials against
a variety of diseases including rheumatoid arthritis, multiple
sclerosis, and Crohn’s disease (7, 8, 9). Their efficacy has helped
establish a set of common effectors in these apparently disparate
diseases. Alternatively, the cytokines whose normal physiological role
is to usher a response from the inflammatory effector phase back to
homeostasis also are being evaluated for their clinical potential as
drugs. The cytokines IL-10 and IL-11 both appear to accelerate this
process and their administration have proven effective in resolving
several animal models of chronic inflammatory disease (10).
Oncostatin M (OM)4 is a pleiotropic cytokine that is produced by activated T cells and macrophages and has shown in vitro properties that would be expected to influence the course of inflammatory responses (11, 12). The protein is structurally and functionally related to IL-6, leukemia inhibitory factor (LIF), and IL-11, proteins that also influence immune and inflammatory function (13). Despite each protein signaling via a family of related receptors and sharing various common properties, each is endowed with a unique array of biological functions (13). Numerous activities have been ascribed to OM in vitro, including the differentiation of megakaryocytes, inhibition of tumor cell growth, induction of neurotrophic peptides, regulation of cholesterol metabolism, and effects on bone-derived cells (7, 14, 15). Recently a collective picture of OM has emerged that strongly suggests a natural role of the cytokine in the wound healing process and attenuation of the inflammatory response. We have previously found that OM can modulate the expression of IL-6, an important regulator of various aspects of the host defense system (16). OM has been shown to regulate the expression by human cells of acute phase proteins and protease inhibitors that have been implicated in modulating cytokine function and limiting tissue damage at sites of inflammation. Recently many of these in vitro effects have been found to occur in rodents and nonhuman primates following OM administration (17). Here we have extended these in vivo findings to further understand the role of OM in regulating cytokine networks following inflammatory stimuli. We have also examined the effects of OM treatment in two murine models of disease in which common proinflammatory cytokines have been previously shown to play key roles.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Studies used female mice (
8 wk old) that were held in
quarantine for 2 wk before admission to any study, during which time
serological examination was performed. BALB/c and C57BL/6 mice were
obtained from Taconic (Germantown, NY), and B10.S-H2(S)SgMcdJ mice were
obtained from The Jackson Laboratory (Bar Harbor, ME). Animals were
housed according to the American Association for the Accreditation of
Laboratory Animal Care and institutional guidelines. Experiments shown
are representative of at least three independent studies. Statistical
analyses were performed using a Wilcoxon rank test (Primer for
Biostatistics, McGraw-Hill, NY).
Reagents
Recombinant human OM was expressed in Chinese hamster ovary
cells and purified as described (18). OM was administered via various
routes of injection in PBS. Escherichia coli LPS (#L3012)
and IFA were purchased from Sigma (St. Louis, MO). TNF- and IL-6
ELISA was obtained from Endogen (Woburn, MA), and no cross reactivity
was found with OM (data not shown). Anti-collagen II monoclonal
hybridomas were purchased from Chondrex (Redmond, WA). Abs were
produced from hybridoma supernatant and purified by protein A Sepharose
chromatography. Mycobacterium tuberculosis was purchased
from Difco (Detroit, MI), and pertussis toxin was obtained from List
Biological Laboratories (Campbell, CA). SRBC were purchased from PML
Microbiologicals (Tualatin, OR), and keyhole limpet hemocyanin (KLH)
was obtained from Pacific BioMarine Lab (Venice, CA). Ab G19-4
(anti-CD3) was provided by Jeff Ledbetter (Bristol-Myers Squibb
Pharmaceutical Research Institute (BMS-PRI), Seattle, WA), mAb 2E12
(anti-CD28) was provided by Bob Mittler (BMS-PRI), and mAb MR1
(anti-murine CD154) was provided by Dr. Tony Siadak (BMS-PRI).
PBMC assays
Human PBMC were prepared from blood obtained from healthy donors by separation on Ficoll. T cells were isolated from this fraction by rosetting with SRBC, and the monocytes were separated from the remaining PBMC by elutriation. T cell populations were >95% CD3+ and monocyte populations were >95% CD14+ as determined by immunostaining. Monocytes were activated by treatment with 5 ng/ml bacterial LPS and T cells were activated by costimulation with immobilized anti-CD3 Ab/soluble anti-CD28 Ab (10 µg/ml). Cells were cultured using RPMI 1640 basal medium supplemented with 10% FBS and penicillin/streptomycin. Culture supernatants were collected at various times following activation for measurement of cytokine content by cytokine-specific ELISA assay.
Cytokine and survival studies
Mice (C57BL/6) were coinjected i.v. with various doses of OM alone or with 1 µg LPS in PBS. OM was injected i.v. at various time points (100 µl, 100 µg/ml) before 1 µg LPS injection, when treatment was delayed. Blood samples were collected via retroorbital sinus into heparinized tubes 1 h after LPS administration. Plasma was removed from the blood following centrifugation and stored frozen at -20°C before assay by ELISA. Control studies showed that comparable cytokine levels were measured in freshly isolated plasma. In survival studies, BALB/c mice were injected with OM (10 µg, 100 µl, 100 µg/ml) i.p. at 4, 2, and 1 h before coinjection of OM and LPS. The final injection contained OM (10 µg) and various doses of LPS in PBS (200 µl). Animals were monitored daily for survival and signs of shock. Moribund animals were sacrificed.
Induction of arthritis by anti-collagen Abs and LPS
Arthritis was induced using the method of Terato et al. (19). BALB/c mice were injected with 400 µl of a mixture of four Abs (D1, D8, A2, F10) in PBS (2.5 mg/ml/mAb, i.v.). At 72 h after mAb injection, mice were injected with LPS (100 µl, 250 µg/ml, i.p.). Treatment with OM (100 µl, 100 µg/ml in PBS, i.v.) or control diluent (100 µl, PBS, i.v.) began 24 h following LPS and continued until day 10. Histopathology and scoring of arthritic disease were adapted from the methods of Wooley et al. (20). Briefly, the extent of disease was scored in a blinded fashion by both visual observation and by measurement of limb swelling with calipers (in 1/1000 of an inch) using the following scale: 0, normal; 1, disease confined to single joints (<80); 2, minimal swelling, minimal redness (90–100); 3, significant swelling, severe reddening, slight foot malformation (100–115); 4, maximal swelling, maximal redness, deformed feet (>115). Measurements of normal animals were in the range 65–75 1/1000th inch. At necropsy, the distal one-third of the limbs were immersion-fixed in formalin, decalcified in HCl, processed by routine methods, and embedded into paraffin. The specimens were sectioned at 4–6 µm, stained with hematoxylin and eosin, and examined by light microscopy. Sections were graded without prior knowledge of the treatment group. Tibiotarsal (hock) joints were graded as to the severity of inflammation, pannus formation, cartilage damage, and osseuos changes. Each parameter was examined separately and graded as follows: grade 0, unremarkable; grade 1, minimal change; grade 2, mild; grade 3, moderate; and grade 4, severe. The inflammation score was derived from evaluation of soft tissue inflammation, synovitis, and angiogenesis. Pannus formation was defined as hypertrophic synovial tissue composed of intraarticular inflammatory exudate accompanied by synovial cell hyperplasia. Cartilage destruction and loss of matrix were evaluated on the articular surfaces of the distal tibia, the talus, the calcaneus, and the tarsal bones to yield the cartilage damage score. The depth of erosion of the subchondral bone and the amount of periosteal exocytosis in the distal tibia, the talus, the calcaneus, and the tarsal bones were evaluated to yield the osseous changes score. The above parameters were then evaluated as to the percent of tissue involved in the disease process: 1, 0–25%; 2, 26–50%; 3, 51–75%; 4, 76–100%. The severity and extent of involvement were then combined to yield the global arthritis score for each joint (maximum possible score, 32).
Peptide synthesis
The peptide 139–151 from proteolipid peptide (PLP) was assembled on a Gilson multiple peptide synthesizer (Middleton, WI) using F-moc amino acids. The peptide resin was treated with trifluoroacetic acid-water-thioanisole-ethanedithiol (100/5/5/2.5) for 2 h, and the cleaved, deprotected peptide was purified by reversed-phase chromatography on a Dynamax C-8 column. The final product was shown to have the expected m.w. by mass spectrometry on a Bio-Ion 20 instrument (Bio-Ion, Uppsala, Sweden).
Induction of experimental autoimmune encephalomyelitis (EAE)
EAE was induced using a protocol similar to that previously described (21). B10.S-H2(S)SgMcdJ mice were immunized by s.c. injection at two sites in the abdominal flanks on day 0 with PLP 139–151 peptide (125 µg) and 300 µg M. tuberculosis H37RA in 200 µl of a 1:1 mixture PBS and IFA. Mice were then injected i.p. with 400 ng pertussis toxin diluted in PBS immediately following the peptide injection. The animals received a second injection of 400 ng pertussis toxin i.p. at 48 h postimmunization. Animals were treated with OM (100 µl, 100 µg/ml, PBS, i.p.) or control diluent (PBS/BSA) on days 4–7 and 12–18. All mice were examined daily for neurological signs of disease. Disease was evaluated as previously described (22) using the following scale: 0, no abnormality; 1, floppy tail with mild hind limb weakness; 2, floppy tail with moderate hind limb weakness; 3, hind limb paresis with or without mild forelimb weakness; 4, hind leg paralysis with or without moderate forelimb weakness; 5, quadriplegia; 6, dead or moribund requiring sacrifice.
Lymph node cell stimulation assay
Animals were immunized with PLP peptide and treated with OM or control diluent as described above. At day 18, animals (5/group) treated with OM or control diluent were sacrificed, and the inguinal and axillary lymph nodes were removed. The nodes from each animal were pooled, a single cell suspension was prepared, and the red cells were lysed. Cells were plated at 500,000 cells per well in media (200 µl, RPMI 1640, 10% FBS, 10 mM HEPES, 50 µM 2-ME) to which was added PLP peptide at various concentrations. Following culture at 37°C for 3 days, proliferation was measured by addition of [3H]thymidine (1 µC/well, 6.7 Ci/mmol; DuPont NEN, Boston, MA) incorporation for 24 h. Media and cells were transferred and washed onto glass fiber filters using a Tomtec cell harvester, air dried, aqueous scintillation fluid was added, and the filters were counted in a Betaplate scintillation counter (LKB Wallac, Gaithersburg, MD).
Immune response to a T cell-dependent Ag
BALB/c mice were injected with 1 x 108 SRBC i.v. (100 µl, PBS) or KLH (250 µg, 100 µl, PBS) i.p. on day 0 then treated with OM (30 µg, i.v., days 0–10), PBS (100 µl, i.v., days 0–10), or anti-CD154 mAb MR1 (23) (200 µg i.v., days 0, 2, and 4). Mice were bled at 7-day intervals and assayed for titers to SRBC or KLH by ELISA as previously described (24).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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OM is produced by both activated T cells and macrophages (11, 16),
therefore we examined the temporal expression of OM in the context of
other cytokines expressed by these cells in response to proinflammatory
stimuli. Treatment of peripheral blood monocytes with E.
coli-derived LPS resulted in a rapid induction of TNF- (Fig. 1
A) (25, 26). Secreted TNF-
levels in the media peaked at 2 h postactivation and then declined
over the next 46 h. Analysis of the same supernatants demonstrated
that OM was produced significantly later than TNF-. Increased OM
levels were first detectable at 24 h post-LPS (>10 pg/ml), then
continued to rise over the next 24 h, reaching a maximum of 1000
pg/ml at 48 h. These results demonstrate that OM secretion from
monocytes is significantly delayed following cell activation with a
pro-inflammatory stimulus. In T cells, induction of IL-2 and other
activation markers rapidly follows receptor-mediated signaling (27). As
shown in Fig. 1B, activation of human peripheral blood T
cells by the cross-linking of Abs to CD28 and CD3 rapidly induced IL-2
to a peak level of 8000 pg/ml at 24 h postactivation, which then
declined to near baseline levels at 72 h. OM was produced
significantly later than IL-2 following T cell activation. OM was first
detectable between 24 and 48 h following cell activation, rising
over the next 48 h reaching peak level of 3000 pg/ml. OM appears
coincident with the decline of IL-2 levels and was further delayed from
the induction of IFN-
(Fig. 1B). Therefore, expression of
OM by the two predominant cell types present at sites of inflammation
occurred significantly later than expression of cytokines most closely
associated with the initiation of inflammation.
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Levels of TNF- correlate with the severity of a variety of
chronic inflammatory diseases including rheumatoid arthritis and
multiple sclerosis, and its action is considered to be central in the
pathogenesis of many of these diseases (5, 28). We initially asked if
TNF- production following challenge of mice with LPS was affected by
OM. Following i.v. injection of LPS (1 µg), control animals had the
expected rapid increase of TNF- with maximal levels of 50 ng/ml
being measured 1 h postinjection. OM inhibited the induction of
TNF- expression in a dose-dependent manner when administered
concurrently with LPS (Fig. 2A). Maximal inhibition of
TNF- (75%) occurred at a dose of 1 µg OM. OM treatment did not
need to be concurrent with LPS, as administration up to 24 h
before LPS was still effective in inhibiting TNF- induction (Fig. 2B). Because IL-6 is regulated in vivo by both OM and
TNF- (17, 25), levels of IL-6 in mice receiving a combination of LPS
and OM were also measured. The combination of LPS and OM produced
levels of IL-6 that were significantly greater than the levels produced
by LPS alone (threefold) or OM alone (100-fold) (Fig. 2A).
This result was not expected, as LPS-induced IL-6 expression is a
result of TNF- production (29) and OM blocked the production of
TNF-. Increased levels of IL-6 diminished rapidly with increased
time between OM and LPS injections. Enhanced levels of IL-6 were only
seen when OM was administered with a delay of less than 2 h (Fig. 2B). If OM preceded LPS by 24 h, the IL-6 levels were
then consistent with the reduced TNF- levels. IL-1 levels were also
measured in these experiments and were not significantly altered by OM
administration (data not shown). We next asked whether the increases in
IL-6 expression seen with OM in combination with LPS were the result of
a synergistic or additive effect between OM and TNF- produced by
LPS. Combinations of OM (10 µg) with TNF- (1 µg or 10 µg)
produced significantly higher levels of IL-6 (more than sevenfold) than
each cytokine alone and at greater levels than would be expected from
just an additive effect (Fig. 2C). In vitro studies using
human or murine macrophages isolated from either peripheral blood or
peritoneal exudates were unable to demonstrate a direct effect of OM on
the production of TNF- or IL-6. Also, macrophages isolated from mice
treated with OM produced similar levels of soluble TNF- following
LPS stimulation in vitro to normal controls (data not shown). These
findings suggest that the effects of OM on TNF- production in vivo
are indirect. The levels of IL-10, another antiinflammatory cytokine
that inhibits TNF- production (10), and the proinflammatory cytokine
IL-1ß were not significantly changed in mice treated with OM and LPS
compared with mice receiving LPS alone (data not shown). In conclusion,
OM is able to inhibit TNF- production while augmenting the normal
feedback loop of IL-6 expression.
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, OM treatment increased the maximum
tolerated dose of LPS in mice twofold when compared with animals
treated with control diluent.
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To further investigate the antiinflammatory properties of OM
following inflammatory stimuli, its effects were studied in an
Ab-induced model of rheumatoid arthritis (19). In this model,
inflammation occurs in the absence of a primary immune response,
allowing one to distinguish between two immunoregulatory pathways,
immune response and inflammation, which are often interdependent and
therefore difficult to separate experimentally. To induce joint
inflammation, animals received a mixture of four mAbs to collagen type
II (1 mg each) followed 72 h later by LPS (25 µg) (19). This
protocol induces a severe arthritis 24 h following LPS injection. OM
treatment was initiated on day 4, after joint inflammation was clearly
established, and continued for 7 days thereafter (10 µg, i.v.). As
shown in Fig. 3A, the severity
of joint inflammation was significantly reduced in OM-treated mice
compared with control animals when assessed for the incidence and
severity of arthritis (20). Nine of 10 control animals were afflicted
with arthritic injury by day 6 and had a median score of 3.8 ±
1.35. In contrast, only one OM-treated animal had macroscopic
evidence of disease with a score >1 and the median arthritic score was
0.4 ± 1. At day 11 postinduction of disease, the animals were
sacrificed, and the rear limbs were subjected to microscopic
examination of disease. Shown in Fig. 3B are representative
histological sections of joints from OM-treated mice and those treated
with control diluent. Histological examination showed that treatment
with OM completely inhibited the influx of inflammatory cells seen in
control animals and prevented the tissue damage associated with a
severe inflammatory reaction. The inflammation and tissue injury was
quantitated. Nine of 10 control animals had severe inflammation and
tissue injury including pannus formation, connective tissue destruction
and erosion of cartlidge and bone, with an average score of
26.9 ± 16.2. The OM-treated mice had histological measures of
inflammation and injury consistent with macroscopic evidence, and this
group had significantly better score of 2.4 ± 7.6
(p < 0.001). In two additional independent
studies, similar efficacy was seen and the cessation of OM treatment at
day 7 was not followed by a delayed onset of inflammation in animals
monitored for an additional 14 days (data not shown).
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To further evaluate the ability of OM to suppress the inflammatory process, its effects were studied in a murine model of multiple sclerosis, EAE. This model shares many inflammatory components that are key to the destruction of the neural sheath and disease progression in multiple sclerosis (30) and to joint destruction in rheumatoid arthritis. In contrast to the arthritis model, T cells are responsible for the inflammatory stimulus in EAE (31). Further, the model also provided a means to independently measure the effects of OM on the immune and inflammatory components of the disease.
Groups of susceptible mice (B10.S-H2 strain) were immunized with a
peptide from a myelin sheath protein, PLP. The peptide contained amino
acids 139–151 of PLP and has been previously demonstrated to be
encephalogenic (32). Immunization with the peptide (in IFA and 3 mg/ml
M. tuberculosis) was followed by two injections with
pertussis toxin as previously described (32). Animals were treated with
OM (10 µg, i.p.) or control diluent i.p. on days 4–7 and days
12–18. At day 11 postimmunization, control animals began to exhibit
neurological symptoms of the disease, particularly paralysis. By day
15, 9 of 10 animals in the control group had succumbed to the disease,
and the median score was 4 (Fig. 4A). In contrast, no animal
that received treatment with OM showed overt signs of the disease in
the 18 days following initiation of the disease-inducing immunization.
Inhibition of the inflammation associated with this disease was
confirmed by histological examination of the brain and spinal cord.
Control animals treated with diluent had lesions typical of EAE (Fig. 4B). The majority of the infiltrate were mononuclear cells
(mainly T cells and smaller numbers of macrophages) and a few
granulocytes. Infiltration involved the meninges with extension in a
perivascular, white matter orientation. In contrast, inflammatory
infiltrate was not detectable in the histology of OM-treated animals.
The extent of damage in the histological examination correlated with
the symptoms of disease when quantitated (OM 0.10 + 0.10, Control 1.60
+ 0.31; p 
0.001). In two additional, independent
studies using other strains of mice with either myelin basic protein or
PLP as the immunogen, OM was comparably effective in blocking the
manifestation of disease (data not shown).
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A). Consistent with this,
there was no significant difference in levels of circulating Abs to the
PLP peptide found in control and OM-treated animals (data not shown).
In parallel studies, we asked if OM treatment comparable to that which
inhibited inflammatory responses modified the immune response to either
of two T cell-dependent Ags, KLH and SRBC (Fig. 5B).
Injection of SRBC elicited a strong immune response with an IgG1 titer
on day 21 (1/3300) consistent with previous findings (24). An Ab to
murine CD154 was included in the study at doses previously shown to be
suppressive (P. M. Wallace, unpublished observations) as a
positive control. No significant difference was seen in animals that
received OM (10 µg) daily for 10 days following immunization.
Similarly, a comparable treatment with OM had no effect on the immune
response to injected KLH. These collective results suggest that
treatment of animals with OM at levels sufficient to inhibit the
inflammatory/degradative aspects of the disease does not suppress
normal Ab production or T cell responsiveness.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, respectively. The role IL-2 may
play in the subsequent induction of OM by T cells was not addressed in
these studies. However the kinetics of cytokine expression are
consistent with the recent findings that the murine OM gene is
inducible by IL-2 (33), and the idea that delayed OM production may
represent a regulatory function involved in a feedback mechanism
following an initial response. The inhibition of endotoxin-induced
TNF- production by OM further supports a role for OM in an
attenuation phase following an inflammatory stimulus rather than in the
initiation or effector phases. The significance of this attenuation was
demonstrated as OM increased the survival of endotoxin-treated animals.
Interestingly, patients with septic shock have elevated OM levels (34).
The efficacy of OM treatment is also demonstrated in murine disease
models of rheumatoid arthritis and multiple sclerosis. Studies of both
rheumatoid arthritis and multiple sclerosis have clearly demonstrated
the importance of TNF- in disease progression, and in models of each
disease a variety of TNF- antagonists have proven efficacious (35).
Therefore, it is reasonable that inhibition of TNF- by OM would
protect from other inflammatory stimuli, such as in autoimmune disease,
in addition to LPS. Others have reported that commercially available
OM, expressed in bacteria, is proinflammatory and induces the
expression of adhesion molecules on endothelial cells in vitro, and an
inflammatory infiltrate in vivo (36). Using highly purified mammalian
protein or yeast derived protein, we have previously studied the
effects of OM on endothelial cells in vitro (16, 37) and injected
protein in vivo in five species (17) (P. M. Wallace,
unpublished observations), with no evidence of these findings.
These differences may reflect differences in the sources and purity of
the proteins used.
The mechanisms by which OM inhibits TNF- remain to be elucidated. No
direct effects of OM on TNF- production could be demonstrated on
macrophages either treated ex vivo or isolated from OM-treated animals.
IL-6 has been found to inhibit TNF- production in vivo (34) and is
induced by OM (17). However, both the inhibition of TNF- by OM in
IL-6-deficient mice (P. Wallace, unpublished results) and the kinetics
of IL-6 induction in the present study make it unlikely that IL-6 is
necessary for TNF- inhibition when LPS and OM are coadministered.
However, IL-6 may participate in the sustained effects of OM when LPS
administration was delayed.
In addition to the inhibition of TNF-, collateral
antiinflammatory properties of OM are likely to contribute to the
effects in these disease models. Cell culture studies have shown that
OM can block and modify the response to IL-1, the agent
provocateur most closely associated with TNF- in mediating tissue
damage at sites of inflammation (38). In vivo, inflammatory
stimuli resulting in production of IL-1 initiate a cascade of effectors
including IL-8 and GM-CSF that amplify the inflammatory response by
recruiting, expanding, and activating inflammatory cells (39). While OM
has no apparent effect on IL-1 production, Richards et al. have
demonstrated that gene expression of GM-CSF and IL-8 induced by
treatment of synovial fibroblasts with IL-1 are suppressed by
cotreatment with OM in a dose-dependent manner (39). One of the primary
chemoattractants of neutrophils, IL-8 also stimulates the production of
neutrophil peroxide and the exocytosis of tissue degradative granules
at sites of inflammation (40). Coincident with suppression of GM-CSF
and IL-8, OM acted synergistically with IL-1 to induce expression of
IL-6 and tissue inhibitor of metalloproteinase-1 (TIMP-1) in these same
cells. Interestingly, the ability of OM to synergize with IL-1 to
suppress inflammatory cytokine expression and induce expression of IL-6
was not paralleled by other members of this cytokine family (39).
OM treatment in vivo may also act indirectly on the function of IL-1
and TNF- by inducing a constellation of protein antagonists. The
acute phase proteins serum amyloid A (SAA) and -1 glycoprotein are
produced locally following tissue injury to minimize damage proximal to
the site of injury. In addition, systemic release of cytokines results
in an acute phase response by the liver to down-regulate the
inflammatory response and reestablish homeostasis. These proteins are
normally produced by adult mammals in response to tissue injury and/or
infection (41) and in a normal, self-limiting process are induced by
IL-1 and TNF- themselves. Administration of -1
glycoprotein protects animals from TNF--induced lethality (42). SAA
and -1 glycoprotein produced during the acute phase are thought to
decrease inflammation by sequestering circulating IL-1 and decreasing
TNF- expression, respectively (22, 43, 44). We have previously
demonstrated that administration of OM can up-regulate the expression
of SAA and -1 glycoprotein in vivo in both mice and in nonhuman
primates (17). Corticosteroids are also potent inhibitors of
proinflammatory cytokines, including IL-1, IL-8, and TNF-. OM in
combination with IL-1 stimulates the hypothalamus-pituitary-adrenal
axis to secrete corticosterone (45), providing an additional mechanism
whereby it can feedback to attenuate inflammation.
Infiltration of inflammatory cells into the articular synovium or the
CNS during acute and chronic inflammation results in tissue damage. The
secretion of reactive oxygen intermediates, and destructive proteases
including neutrophil elastase, cathepsins, and matrix
metalloproteinases by these activated cells degrade connective tissue
and cartilage in rheumatoid arthritis and the neural sheath in multiple
sclerosis (46). In addition to attenuating the cytokines that stimulate
secretion of these proteases, in vitro studies have demonstrated that
OM is capable of inducing a spectrum of protease inhibitors. The acute
phase proteins induced by OM also include two major serine proteinase
inhibitors: 1-proteinase inhibitor (1-Pi) and
anti-chymotrypsin. 1-Pi, the primary inhibitor of neutrophil
elastase, is secreted from lung epithelial cells and synovial
fibroblasts stimulated by OM (C. Richards, unpublished observations).
In comparison, LIF and IL-6 have little or no effect on expression of
1-Pi. Interestingly, the stimulation of 1-Pi by OM is greatly
enhanced in the presence of IL-1. Anti-chymotrypsin, an inhibitor of
cathepsin G and other chymotrypsin-like enzymes secreted during
inflammation, also inhibits superoxide generation by activated
neutrophils (47). Anti-chymotrypsin is produced following OM treatment
of hepatic and numerous nonhepatic human cells (48). Its expression by
epithelial cells in response to treatment with OM is synergistically
increased by cotreatment with OM and corticosteroid (48).
In vivo, the action of collagenase and gelatinase are regulated by the
relative level of their cognate inhibitor, TIMP-1 (49). OM increased
the expression of TIMP-1 by synovial fibroblast and did so more
effectively than other members of the IL-6 cytokine family (50).
Similarly, OM stimulated the production of TIMP-1 from human articular
chondrocytes and cartilage explant culture more effectively than IL-11,
LIF, or IL-6 (51). Expression of TIMP-1 from fibroblasts in response to
OM occurs with no effect on matrix metalloproteinase (MMP) levels,
resulting in a net decrease in MMP activity (50). The effects of OM on
1-Pi, anti-chymotrypsin, and TIMP-1 expression are greatly
enhanced in the presence of IL-1 (52), again suggesting that OM works
in consort with proinflammatory molecules as part of an
antiinflammatory feedback loop. Based on the numerous cell types
responsive to OM, it is reasonable to expect this synergistic feedback
could occur at sites of inflammation.
The synergy of OM with proinflammatory mediators is also seen for the
induction of IL-6. Although the role of IL-6 in inflammation remains
controversial (53), in vivo IL-6 induces IL-1 receptor antagonist and
soluble TNF receptor p55 that attenuate inflammation (54). IL-6 is a
key inducer of protease- and cytokine-inhibitors that reduce
inflammation and initiate healing. Its protective effects are also
inferred from the failure of IL-6-deficient mice to repair tissue and
recover from inflammation, to attenuate TNF-, or produce proteins
that limit damage at sites of injury (55). In this light, the ability
of OM to enhance IL-6 production is consistent with a role for the
protein in tissue repair. The synergistic interaction of OM and TNF-
on increased IL-6 expression supports the concept that OM activity is
enhanced in the presence of this proinflammatory cytokine at sites of
injury and inflammation. This synergy between pro- and antiinflammatory
cytokines is not unique to OM and TNF-, as OM combined with IL-1
also yields a similar enhancement of IL-6 (45). As described above, the
production of acute phase proteins is also maximized by the combined
presence of OM and inflammatory mediators.
We have demonstrated that expression of OM from activated T cells and
macrophages is temporally delayed and increases coincident with a
decline in expression of TNF- and IL-2. We, and others, have
demonstrated synergy between OM and inflammatory cytokines in
suppression of inflammatory mediators, and we have shown herein that,
administered systemically, the molecule is efficacious in three
different models of acute disease with common proinflammatory
mediators. The recent cloning of the murine OM-specific receptor has
called into question the interaction of the human protein in murine
studies (56). However, cytokine production and the generation of acute
phase proteins also occur from human cells treated with the human
protein. Similarly, we have also established that OM functions in
nonhuman primates to inhibit LPS-induced TNF- production and
up-regulate both IL-6 (P. M. Wallace and A. F. Wahl,
unpublished observations) and acute phase proteins (17). In the context
of an inflammatory cycle, initiators such as TNF- and IL-1, which
promote inflammatory cell activation and secretion of chemoattractants
and proteinases, would remain maximal at the peak of inflammatory
response. Local expression of OM in the presence of these activators
would then potentiate a return to homeostasis as the proinflammatory
mediators are suppressed.
Taken together, these in vivo findings suggest that OM participates in
attenuating the inflammatory responses and restoring normal homeostasis
following tissue injury and/or infection. The ability of OM to enhance
the negative feedback of proinflammatory cytokine production, in
addition to inhibiting their biological effects, distinguishes the
therapeutic potential of this molecule from those of individual
cytokine antagonists such as IL-1 receptor antagonist or
anti-TNF- soluble receptor. Moreover, some key antiinflammatory
activities of OM such as its direct effect on fibroblasts and
epithelial cells and its induction of protease inhibitors are not seen
with other antiinflammatory cytokines such as IL-6, IL-10, or IL-11.
Such OM-specific responses may reflect the tissue distribution of the
recently characterized OM-specific receptor complex being coupled to a
specific set of OM-inducible genes (57). These data indicate OM could
act therapeutically in the treatment of inflammatory diseases by
regulating the spectrum of events that comprise a natural feedback loop
to return active inflammation to homeostasis.
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Acknowledgments |
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Footnotes |
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2 Current address: Xcyte Therapies, 2203 Airport Way South, Suite 300, Seattle, WA 98134.
3 Current address: Seattle Genetics, 22215, 26th Avenue SE, Bothell, WA 98021.
4 Abbreviations used in this paper: OM, oncostatin M; LIF, leukemia inhibitory factor; 1-Pi, 1-proteinase inhibitor; EAE, experimental autoimmune encephalomyelitis; KLH, keyhole limpet hemocyanin; MMP, matrix metalloproteinase; PLP, proteolipid protein; SAA, serum amyloid A; TIMP-1, tissue inhibitor of metalloproteinase-1; BMS-PRI, Bristol-Myers Squibb Pharmaceutical Research Institute.
Received for publication November 19, 1998. Accepted for publication February 16, 1999.
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