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*
Department of Neurology, Vanderbilt University Medical Center, Nashville, TN 37212; and
Cell Therapeutics, Inc., Seattle, WA 98119
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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production ex vivo after Ag restimulation. The inhibition of Th1
differentiation in vivo is consistent with a block in IL-12 receptor
signaling, because LSF blocked IL-12-driven Th1 differentiation and T
cell proliferation in vitro, yet had no effect on IL-12 secretion from
APCs ex vivo or in vitro. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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) are detected in the CNS at the height of
disease in both humans with MS and mice with EAE (2, 3, 4), and,
conversely, neutralizing cytokine-specific Abs ameliorate disease
progression in the murine model (5, 6, 7). Because Th2-type cytokines are
predominantly detected in murine brain during recovery (8), and because
disease is suppressed by administration of Th2-type cytokines (9, 10),
the data suggest that there is a switch from a Th1-type response to a
Th2-type response during remission.
IL-12 is a 70-kDa heterodimeric cytokine produced by macrophages in
response to infection, LPS, CD154 (CD40 ligand), and IFN- (11).
IL-12 is critical for the development of Th1-type cells and influences
Th1 responses primarily by inducing IFN- production from T cells and
NK cells (12). A lack of IL-12 signaling leads to almost complete
absence of Th1 cells and reduced IFN- production (13, 14). The
importance of IL-12 has been extended to the pathogenesis of Th1
cell-mediated autoimmune disease. Prevention of EAE and
trinitrobenzenesulfonic acid-induced murine colitis by neutralizing Abs
against IL-12 and augmentation of diabetes in NOD mice by exogenous
IL-12 have suggested an important role for IL-12 in the pathogenesis of
these diseases (5, 15, 16). IL-12 neutralizing Abs also inhibit
Th1-mediated acute graft-vs-host disease (17).
Lisofylline (LSF), 1-(5-R-hydroxyhexyl)-3,7-dimethylxanthine, is a novel anti-inflammatory compound that abrogates release of proinflammatory cytokines during oxidative lung injury (18), reduces inflammatory cytokine release in response to cytotoxic cancer chemotherapy (19), and protects minipigs from sepsis (20). In clinical trials involving patients receiving sibling donor, HLA-matched bone marrow transplants (BMT) after ablative chemotherapy and radiation therapy, LSF recipients experienced a significant reduction in neutropenic infection and treatment-related mortality and had improved survival (21). Reduction in toxicity and improved survival correlated with lower levels of oxidized lipids in sera from the patients (22). From these and other data, a regulatory paradigm is suggested in which inflammatory cytokines induce species of oxidized lipids (23), which, in turn, leads to amplification of inflammatory cytokine secretion (24, 25). Based on the anti-inflammatory properties of LSF in multiple models of acute inflammation, we employed LSF to inhibit the induction of EAE and examined its mechanism of action in this inflammatory, Th1-mediated autoimmune disease.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Female SJL/J mice were purchased from Clarence Reader (National
Institutes of Health, Bethesda, MD), and BALB/c mice were purchased
from Charles River Labs (B & K Universal, Fremont, CA); SJL/J and
BALB/c mice were maintained at Vanderbilt University Medical Center and
Cell Therapeutics, Inc., respectively. Anti-IL-12 (C17.5 and C15.6)
were a kind gift of Dr. G. Trinchieri (Wistar Institute, Philadelphia,
PA). The Abs RA3-3A1/6.1 (anti-B220), J11d.2, and MAR18.5
(anti-rat 
-chain) were purified from culture supernatants;
anti-CD3
(145-2C11), anti-mouse IFN-
(XMG1.2) and
anti-CD40 (HM40-3) were purchased from PharMingen (San
Diego, CA), and anti-IFN- Ab (MM700) was obtained from Endogen
(Woburn, MA). Recombinant murine IL-12 and IL-4 was purchased from
Genzyme (Cambridge, MA), recombinant human IL-10 (active on murine
cells) was purchased from PeproTech (Rocky Hill, NJ), guinea pig
complement was purchased from Accurate Chemical (Westbury, NY), and
recombinant murine IFN- was purchased from Biosource International
(Camarillo, CA). LPS and pentoxifylline (PTX) were obtained from Sigma
(St. Louis, MO).
Induction of EAE
Active EAE was induced by immunization of SJL/J mice with 800 µg of mouse spinal cord homogenate (MSCH) (10) in CFA on days 0 and 7. Passive EAE was induced by adoptive transfer of myelin basic protein (MBP)-sensitized T lymphocytes as follows: SJL/J mice (4- to 6-wk-old) were immunized on days 0 and 7 with 400 µg of MBP in IFA containing 50 µg/ml of H37RA. On day 14, the regional draining lymph node cells (LNC) and spleen were harvested and pooled and cultured at 4 x 106 cells/well in RPMI 1640 containing 10% FBS (HyClone Labs, Logan, UT), 2 mM L-glutamine, 5 x 10-5 M 2-ME, 1% penicillin/streptomycin, and 100 µg/ml of MBP. After 4 days, viable T cell blasts were harvested by Ficoll centrifugation, washed twice in PBS, and injected i.p. into recipient mice (1.0 x 107 to 1.5 x 107 cells in 500 µl of PBS).
Treatment of animals with LSF and development of clinical and pathologic scores
Mice were given either LSF (2 mg/ml in PBS) or PBS twice daily by gavage needle in volumes of 0.25 or 0.5 ml/dose. Paralysis was graded as follows; 0, no paralysis; 0.5, stiff tail; 1, limp tail; 1.5, limp tail with inability to right; 2, paralysis of one limb; 2.5, paralysis of one limb and weakness of one other limb; 3, complete paralysis of both hind limbs; 4, moribund state; 5, death. To assess the degree of inflammation, mice were euthanized on day 25 and perfused by intracardiac injection of 4% paraformaldehyde and 1% glutaraldehyde in PBS. Transverse sections of the cervical, upper thoracic, lower thoracic, and lumbar region of the spinal cord were stained with Luxol Fast Blue or with hematoxylin and eosin. Each spinal cord section was further subdivided into an anterior, posterior and two lateral columns, and sections displaying either lymphocyte infiltration or demyelination were assigned a score of one. Thus, each animal had a potential maximum score of 16.
Proliferation assay
Fifteen days after MBP immunization, PBS- or LSF-treated donor cells were cultured at 2 x 105/well with MBP for 72 h, and pulsed during the last 18 h with [3H]thymidine (0.5 µCi/well). [3H]Thymidine incorporation was measured on a betaplate liquid scintillation counter (Wallac, Turku, Finland).
Cells and IL-12 secretion
Normal macrophages were enriched from single-cell suspensions of BALB/c spleens by adherence to tissue culture plates at 37°C for 1 h. Plates were rinsed four times with PBS before harvesting the adherent cells. Normal macrophage or the murine macrophage cell line J774A.1 (American Type Culture Collection No. TIB-67; Manassas, VA) were incubated for 48 h as indicated, and the culture media were collected and assayed for IL-12 by ELISA.
Induction of Th1 differentiation
T cells were enriched (>95%) from RBC-depleted BALB/c
splenocytes by complement-mediated lysis of B cells using rat
anti-mouse B220 and J11d, followed by cross-linking with MAR18.5.
Anti-CD3 (4 µg/ml in PBS) was immobilized to tissue culture plates by
incubation for 1 h at 37°C. After the plates were washed twice
in PBS, T cells (0.5 x 106 cells/ml) were stimulated
in the presence or absence of IL-12, with or without LSF or PTX. After
7 days, equal numbers of viable cells were restimulated overnight at
5 x 105 cells/ml with insoluble anti-CD3, and
supernatants were collected and assayed for IFN-.
ELISA for IL-12, IFN-, and IL-4
IL-12 secretion was determined by a sandwich ELISA. Plates were
coated with 2 µg/ml of anti-IL-12 (C17.15) and blocked with 3%
BSA in PBS. After overnight incubation at 4°C, sample wells were
washed with PBS/0.05% Tween 20, and incubated with biotinylated
anti-IL-12 (C17.5) at 0.2 µg/ml. After 1 h at room
temperature, the plates were washed, incubated for 1 h with
avidin-alkaline phosphatase, and washed again before addition of 1
mg/ml of p-nitrophenyl phosphate. Absorbance was read at 405
nm. The IL-12 concentration was calculated by interpolation from a
standard curve. IFN- production was also measured by a sandwich
ELISA as described above, except for using R4-6A2 as the capture Ab and
biotinylated MM700 as the second step. Total IL-12 and IFN- levels
were assayed with Intertest-12 and Intertest- ELISA kit,
respectively, both also Genzyme (Cambridge, MA).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Mice were immunized with MSCH in CFA to induce active EAE, and
treated with either LSF (1 mg per dose i.e., 50 mg/kg; see inset, Fig. 1
) or PBS by gavage twice daily for 15
days; the summed data from two separate experiments are shown (Fig. 1).
In control groups, 7 of 10 animals receiving PBS developed hind limb
paralysis with a mean clinical score of 2.4 on day 20. In contrast,
only 2 of 13 animals receiving LSF became paralyzed and the mean
clinical score was 0.75 (p < 0.025). No
significant difference in the day of onset of paralysis was seen. LSF
also prevented the development of passive EAE induced by adoptive
transfer of MBP-sensitized T cells. All six mice receiving PBS
developed paralytic signs with a mean clinical score of 2.35 on day 9
(Fig. 2). Mice given LSF at either 0.5 or
1.0 mg/dose twice a day were significantly protected, and only 1 of 12
animals that received LSF developed hind limb paralysis
(p < 0.025).
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, A and B).
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Because LSF decreased the severity of paralysis in both models of
EAE, we examined the effects of LSF treatment in vivo on the potency
and phenotype of MBP-reactive T cells. Mice immunized with MBP in CFA
were given either LSF (0.5 mg/dose) or PBS twice daily for 14 days. On
the fifteenth day, spleen and LNC were pooled and cultured in vitro
with MBP for 4 days. At each day of the 4-day in vitro culture with
MBP, a sample of culture supernatant was collected and tested for the
amount of IFN- and IL-4 produced, and after 4 days blast cells from
each group were compared for their potency at adoptively transferring
EAE. Duplicate cultures were also tested for proliferative capacity in
response to MBP.
When compared with cells from PBS-treated mice, donor cells from
LSF-treated animals were less effective at inducing passive EAE after
transfer into naive hosts. In experiment 1 (Fig. 4A), all six animals that
received PBS-treated donor cells developed clinical paralysis (mean
clinical score = 4.5), and four died at the height of disease. In
contrast, none of the mice that received cells from LSF-treated mice
died, the mean maximum score was 3.8, and all animals recovered to a
mean score of 1.8 by day 24. In the second experiment, the clinical
severity in mice receiving PBS-treated cells was 3.25, whereas in the
LSF group it was 2.0 (Fig. 4B). The reduced
encephalitogenicity of MBP-specific T cells that were generated in
LSF-treated mice was long lasting, as these T cells were transferred to
naive recipients without any additional drug administration. The
reduction in clinical severity of EAE induced by LSF-treated donor
cells vs that induced with PBS-treated donor cells was significant
(p < 0.001) and was not due to differences in
lymphocyte activation, because both populations proliferated equally
well in response to MBP in vitro (Fig. 4C).
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at each of the 4 days tested (Fig. 4
D). The culture supernatants also displayed reduced IL-4
production up to day 3, although the relative inhibition of IL-4
production by ex vivo-activated T cells from LSF-treated mice was not
as great as that observed for IFN- (Fig. 4E). Effect of LSF on IL-12 secretion and signaling
The previous experiments suggested that LSF inhibited Th1
differentiation in vivo, as shown by a reduced capacity to induce EAE
and secrete IFN- ex vivo in response to Ag. Because IL-12 is a key
cytokine that regulates Th1 differentiation, the lack of Th1 cells
could arise from a block in IL-12 secretion from APC or an inhibition
of IL-12 signaling in T cells. To test the former possibility, we
measured total IL-12 production from macrophages activated in vitro in
the presence of LSF using the maximal concentrations detectable from
patients’ sera during clinical trials and up to fourfold higher.
Normal murine macrophages were induced to secrete IL-12 after
activation with anti-CD40 or LPS (Fig. 5A), but this effect was not
inhibited by LSF; even at concentrations of anti-CD40 inducing
suboptimal IL-12 production, LSF was not inhibitory. Furthermore, LSF
did not block IL-12 secretion from a murine macrophage line stimulated
with LPS and IFN- (Fig. 5B), whereas IL-10 completely
abolished IL-12 secretion.
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upon
secondary stimulation (Fig. 6A). Strikingly, the presence
of LSF during T cell differentiation inhibited Th1 maturation in a
dose-dependent fashion, such that the amount of IFN- secreted was
equivalent to that of T cells activated with anti-CD3 alone, in the
absence of IL-12 (Fig. 6A). A similar pattern of inhibition
was seen when culture supernatants were tested for the production of
other Th1 cytokines; secretion of IL-2 and TNF- by T cells
responding to IL-12 in the presence of LSF were also inhibited with
respective IC50 (concentration of drug where 50%
inhibition of response occurs) values of 15 µM and 20 µM (data not
shown). Similar LSF concentrations also inhibited Th1 differentiation
induced by addition of heat-killed Listeria monocytogenes
(data not shown), which enhances Th1 development by inducing IL-12
secretion from macrophages. LSF did not affect the viability or
recovery of T cells after 1 wk of culture (data not shown), consistent
with its inability to block lymphocyte activation (Fig. 4C).
In support of this, Th1 differentiation was also inhibited even when
both LSF and IL-12 were added up to 48 h after anti-CD3
activation, indicating that LSF blocked the response to IL-12 and not
the acquisition of IL-12 responsiveness (data not shown). LSF also had
little effect on the low levels of IFN- secreted from T cells
stimulated with anti-CD3 alone, confirming it does not induce
anergy or TCR desensitization (Fig. 6A). Furthermore, LSF
did not augment Th2 differentiation at the expense of Th1 development,
as shown above for MBP-specific T cells activated ex vivo (Fig. 4E), because the levels of IL-4 and IL-10 secretion detected
in these cultures were not enhanced (data not shown).
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A, LSF inhibited IL-12-induced Th1
differentiation in a dose-dependent fashion, resulting in a >85%
blockade at 50 µM (Fig. 6B). Th1 differentiation in the
presence of LSF was approximately reduced to levels seen when T cells
were stimulated with anti-CD3 alone in the absence of exogenous
IL-12 (5.8 ng/ml). In contrast, equimolar concentrations of PTX had no
effect on IL-12-induced Th1 differentiation. The stereochemical
enantiomer of LSF, 1501S, also had no effect on Th1 maturation (data
not shown).
Although the inhibition of IL-12-induced Th1 maturation by LSF did not
lead to augmented Th2 differentiation (both in vivo and in vitro), it
was unclear whether LSF might block Th2 differentiation in vitro driven
by exogenously added IL-4. As expected, anti-CD3-stimulated T cells
coincubated with IL-4 displayed increased Th2 differentiation as
compared with T cells activated with anti-CD3 alone (Fig. 6C). Addition of LSF did block IL-4-induced Th2
differentiation, yet the percent inhibition observed was less than that
for Th1 differentiation driven by the addition of exogenous
IL-12.
Because LSF inhibited IL-12-driven Th1 differentiation both in vivo and in vitro, we examined whether LSF was capable of inhibiting additional IL-12-dependent responses in vitro. In addition to its effects on Th1 differentiation, IL-12 is a T cell growth factor that augments the proliferation of T cell blasts (11). Therefore, we tested, the ability of LSF to block proliferation of T cell blasts in response to IL-12. In preliminary experiments, we determined that the optimal T cell response to IL-12 occurred 3 days after T cell stimulation with anti-TCR reagents or mitogens (data not shown).
Splenic T cells stimulated for 3 days with Con A showed a
dose-dependent increase in IL-12-mediated proliferation, which was
optimal between 5 and 25 U/ml of IL-12 (Fig. 7, A and B). T cell
blasts coincubated in the presence of LSF displayed reduced
proliferative responses to IL-12, resulting in a dose-dependent
decrease in T cell growth, even when optimal IL-12 concentrations were
used (Fig. 7A). Importantly, LSF had no inhibitory effect
when T cells were activated for 3 days in the presence of LSF, i.e.,
during the acquisition of IL-12 responsiveness, provided the LSF was
washed out before addition of IL-12 (Fig. 7B). Therefore,
the presence of LSF had no effect on T cell activation or the ability
to induce IL-12 responsiveness, but rather LSF blocked the ability of T
cell blasts to respond to IL-12.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and 2). LSF-treated mice were protected from lymphocyte
infiltration into neural tissues and demyelination, as demonstrated
histologically by analysis of spinal cord sections (Fig. 3). Thus, LSF
blocked progression of an inflammatory, Th1-mediated autoimmune
disease.
To determine how LSF ameliorates disease progression, we examined ex
vivo proliferative responses of Ag-specific T cells from LSF-treated
mice and their profiles of cytokine secretion. LSF administration in
vivo did not inhibit the ex vivo T cell proliferative response to MBP
(Fig. 4C), indicating that LSF does not induce tolerance,
block Ag-specific priming, or inhibit the cellular response to
subsequent Ag challenge. This is consistent with the inability of LSF
to inhibit expression of T cell activation Ag, IL-2 secretion, a
mitogenic response, or proliferation in response to anti-CD3 plus
or minus anti-CD28 costimulation in vitro (data not shown).
Furthermore, in vivo treatment with LSF did not diminish IL-12
secretion by MBP-primed APC ex vivo (data not shown), suggesting that
LSF did not affect APC function and cytokine secretion.
A striking correlation emerged when we analyzed the Th cell phenotype
and encephalitogenicity of donor cells from LSF-treated mice. LSF
treatment in vivo reduced the amount of IFN- secreted by ex vivo T
cells stimulated with Ag (Fig. 4D), implying that Th1
differentiation had been inhibited in vivo. LSF inhibited Th1
differentiation by blocking IL-12 signaling and not by inhibiting IL-12
secretion from APC (Fig. 5), because IL-12-dependent Th1
differentiation was suppressed in vitro (Fig. 6, A and
B). We extended these observations to show that LSF
abrogates IL-12-induced proliferation of anti-CD3-activated T cell
blasts (Fig. 7A), but not the ability to induce IL-12
responsiveness during T cell activation (Fig. 7B). The
reduced capacity to produce IFN- in vitro correlated with a reduced
capacity to adoptively transfer EAE to susceptible, naive recipients,
indicating that the inability of T cells from LSF-treated mice to
transfer disease was a long-lived effect and suggesting that it stemmed
from an abrogation of Th1 effector function. Thus, donor cells from
LSF-treated animals were less encephalitogenic, which led ultimately to
lower clinical scores (Fig. 4, A and B).
IL-12 induces IFN- production by NK cells and T cells, and drives
the differentiation of inflammatory Th1 cells. IL-12 was detected in
early inflammatory lesions in MS brains and correlated with APC CD80
expression (3). We have shown that IL-12 expression levels in mouse
brain and spinal cord correlate with the development of paralytic signs
in EAE (29). Because anti-IL-12 abrogates development of CNS
demyelination and induction of EAE (5, 30), the inhibition of IL-12
signaling by LSF, which leads to reduced Th1 differentiation, is
consistent with its modulatory effect on the pathobiology of this
model. LSF did not appear to act by shifting the balance of Th cell
differentiation toward a Th2 phenotype, which has been shown to be
ameliorative in EAE (8, 9, 10). Indeed, analysis of supernatants derived
from activated T cells treated with LSF in vivo (Fig. 4E) or
in vitro (data not shown) did not detect enhanced secretion of IL-4.
Several studies document a correlation between the presence of Th1
cytokines in MS and in experimental models of CNS inflammation (2, 4, 6, 7, 31). Although IFN- is the prototypical cytokine associated
with Th1 responses, the role of IFN- in EAE and MS is equivocal.
CSF-derived T cells from MS patients are Th1 cells that secrete
enhanced levels of IFN- (56, 57), and IFN- dosing worsens
disease progression (32). However, IFN- knockout mice remain
susceptible to EAE (33), anti-IFN- may exacerbate disease (34),
and in certain cases IFN- administration is protective (35). These
conflicting results may relate to the effects of IFN- during
development, its differential inflammatory effects systemically and
locally (36), or a compensatory mechanism that leads to overexpression
of other Th1 cytokines which affect EAE pathogenesis (7). Indeed,
organ-specific overexpression of IFN- leads to spontaneous CNS
demyelination, confirming that IFN- is a candidate cytokine for
affecting MS pathogenesis (37). It was recently shown that IFN-
knockout mice remain susceptible to EAE via an IL-12-dependent
mechanism that antagonizes IL-10 production (30). Our data suggest that
LSF affects this immunoregulatory circuit and may provide protection as
a prophylactic in Th1-mediated autoimmune disease by short-circuiting
an inflammatory cascade initiated by IL-12.
The etiology of autoimmune demyelinating disease may directly relate to
IFN- production, because IFN- exacerbates inflammatory responses
in several ways. These include enhanced adhesion of lymphocytes to CNS
endothelium (38), increased permeability of the blood-brain barrier
(31, 39), and augmented MHC expression and Ag presentation (40, 41, 42, 43).
Thus, IFN- coordinately promotes lymphocyte access to the CNS and
presentation of autoantigen by astrocytes and microglia, which are
normally MHC class II negative. IFN- is also the most potent
cytokine for up-regulation of inducible nitric oxide synthase (iNOS) in
macrophages (44). In this regard, iNOS levels and enzyme activity
correspond to disease activity in EAE (45) and correlate with recent
findings linking the levels of peroxynitrates with severity of EAE and
MS (46). It is noteworthy that inflammatory cytokines also induce lipid
peroxidation (23), because peroxidized lipids are evident in MS
patients (47, 48) and the levels of peroxidized lipids in patients
undergoing treatment regimens for BMT are abrogated by LSF
(22).
The ability of LSF to inhibit IL-12-induced Th1 differentiation and, to
a lesser extent, IL-4-induced Th2 differentiation in vitro (Fig. 6C) was not entirely unexpected, because LSF was not
developed as a specific inhibitor of IL-12 signaling pathways. LSF may
act as an inhibitor of STAT activation, because the induction of STAT 4
and STAT 5 tyrosine phosphorylation in response to IL-12 and IL-2,
respectively, are blocked by LSF pretreatment (S.K., manuscript in
preparation). In this regard, it is noteworthy that LSF does block
IL-12-induced T cell proliferation but does not block IL-2-induced T
cell proliferation nor IL-4-induced B cell proliferation (data not
shown), which are thought to proceed via STAT-independent mechanisms
(49, 50, 51). However, the inhibition of EAE by LSF is consistent with a
dominant block in Th1 differentiation, because EAE is a prototypical
Th1-mediated disease, and there is no evidence to support a role for
Th2 cells in this disease. Together, the results suggest that LSF may
prove beneficial in highly polarized Th1 or Th2 responses, where the
production of a predominant set of cytokines induce disease via
STAT-dependent pathways. The effects of LSF on Th2 differentiation in
vivo are currently being examined.
Although PTX is a structural analogue of LSF that also blocks EAE (27),
PTX was not well tolerated in humans receiving BMT and did not prevent
regimen related toxicities (52, 53, 54). Furthermore, concentrations of PTX
routinely used in vitro to inhibit proinflammatory cytokine secretion
or other biologic responses are usually in vast excess of those
tolerated in human patients during clinical trials. Indeed,
administration of PTX in a pilot study of MS patients showed no
evidence for a decrease of Th1 function or Th1-derived cytokines in
stimulated peripheral blood cells (55). In the present study, LSF
inhibited Th1 differentiation in response to IL-12 (Fig. 6B)
at concentrations comparable to those detected in clinical trial
patients undergoing BMT (Cmax = 19 µM); in contrast, PTX
was ineffective. Furthermore, the dose of LSF used to inhibit EAE in
mice (25 or 50 mg/kg) is comparable to that used in human clinical
trials (3 mg/kg) based upon a calculated 15% oral bioavailablity of
LSF in mice (data not shown). With regard to inhibition of IL-12
signaling, LSF was also selectively inhibitory when compared with its
stereospecific S-enantiomer (data not shown), suggesting
that the efficacy of LSF requires the R-chiral alcohol
within the alkyl side chain, a determinant of the pharmacophore. Taken
together, the data suggest that an orally active LSF analogue may
provide a novel therapeutic approach to Th1-mediated diseases such as
MS.
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Acknowledgments |
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Footnotes |
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2 J.J.B. and C.D. contributed equally to this work.
3 Address correspondence and reprint requests to Dr. Stephen J. Klaus, Cell Therapeutics, Inc., 201 Elliott Ave. West, Seattle, WA 98119. E-mail address:
4 Abbreviations used in this paper: EAE, experimental allergic encephalomyelitis; MS, multiple sclerosis; LSF, lisofylline; PTX, pentoxifylline; MSCH, mouse spinal cord homogenate; MBP, myelin basic protein; CNS, central nervous system; BMT, bone marrow transplant; LNC, lymph node cells.
Received for publication April 28, 1998. Accepted for publication August 11, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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H. P. M. Brok, M. van Meurs, E. Blezer, A. Schantz, D. Peritt, G. Treacy, J. D. Laman, J. Bauer, and B. A. 't Hart Prevention of Experimental Autoimmune Encephalomyelitis in Common Marmosets Using an Anti-IL-12p40 Monoclonal Antibody J. Immunol., December 1, 2002; 169(11): 6554 - 6563. [Abstract] [Full Text] [PDF]
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C. Natarajan and J. J. Bright Curcumin Inhibits Experimental Allergic Encephalomyelitis by Blocking IL-12 Signaling Through Janus Kinase-STAT Pathway in T Lymphocytes J. Immunol., June 15, 2002; 168(12): 6506 - 6513. [Abstract] [Full Text] [PDF]
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 M. Chen, Z. Yang, R. Wu, and J. L. Nadler Lisofylline, a Novel Antiinflammatory Agent, Protects Pancreatic {beta}-Cells from Proinflammatory Cytokine Damage by Promoting Mitochondrial Metabolism Endocrinology, June 1, 2002; 143(6): 2341 - 2348. [Abstract] [Full Text] [PDF]
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G.-X. Zhang, H. Xu, M. Kishi, D. Calida, and A. Rostami The Role of IL-12 in the Induction of Intravenous Tolerance in Experimental Autoimmune Encephalomyelitis J. Immunol., March 1, 2002; 168(5): 2501 - 2507. [Abstract] [Full Text] [PDF]
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 A. K. Singh, M. T. Wilson, S. Hong, D. Olivares-Villagomez, C. Du, A. K. Stanic, S. Joyce, S. Sriram, Y. Koezuka, and L. Van Kaer Natural Killer T Cell Activation Protects Mice Against Experimental Autoimmune Encephalomyelitis J. Exp. Med., December 17, 2001; 194(12): 1801 - 1811. [Abstract] [Full Text] [PDF]
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C. Du, M. W. Khalil, and S. Sriram Administration of Dehydroepiandrosterone Suppresses Experimental Allergic Encephalomyelitis in SJL/J Mice J. Immunol., December 15, 2001; 167(12): 7094 - 7101. [Abstract] [Full Text] [PDF]
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 C. Du, Q. Guan, M. W. Khalil, and S. Sriram Stimulation of Th2 Response by High Doses of Dehydroepiandrosterone in KLH-Primed Splenocytes Experimental Biology and Medicine, December 1, 2001; 226(11): 1051 - 1060. [Abstract] [Full Text] [PDF]
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S. Lassmann, C. Kincaid, V. C. Asensio, and I. L. Campbell Induction of Type 1 Immune Pathology in the Brain Following Immunization Without Central Nervous System Autoantigen in Transgenic Mice With Astrocyte-Targeted Expression of IL-12 J. Immunol., November 1, 2001; 167(9): 5485 - 5493. [Abstract] [Full Text] [PDF]
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C. M. Fleming, H. He, A. Ciota, D. Perkins, and P. W. Finn Administration of Pentoxifylline During Allergen Sensitization Dissociates Pulmonary Allergic Inflammation from Airway Hyperresponsiveness J. Immunol., August 1, 2001; 167(3): 1703 - 1711. [Abstract] [Full Text] [PDF]
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 E. Laurat, B. Poirier, E. Tupin, G. Caligiuri, G.K. Hansson, J. Bariety, and A. Nicoletti In Vivo Downregulation of T Helper Cell 1 Immune Responses Reduces Atherogenesis in Apolipoprotein E-Knockout Mice Circulation, July 10, 2001; 104(2): 197 - 202. [Abstract] [Full Text] [PDF]
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M. E. Coon, M. Diegel, N. Leshinsky, and S. J. Klaus Selective Pharmacologic Inhibition of Murine and Human IL-12-Dependent Th1 Differentiation and IL-12 Signaling J. Immunol., December 15, 1999; 163(12): 6567 - 6574. [Abstract] [Full Text] [PDF]
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 C. L. S. George, G. Fantuzzi, S. Bursten, L. Leer, and E. Abraham Effects of lisofylline on hyperoxia-induced lung injury Am J Physiol Lung Cell Mol Physiol, May 1, 1999; 276(5): L776 - L785. [Abstract] [Full Text] [PDF]
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